Antagonistic CD40 monoclonal antibodies and uses in treating immune responses

ABSTRACT

The disclosure provides for antibodies that bind CD40, including a humanized antibody. The antibodies bind CD40 and do not exhibit CD40 agonist activity. The antibodies may comprise a modified IgG1 Fc domain, and exhibit minimal activation of immature dendritic cells. Compositions comprising antibodies, methods of use for treatment of diseases involving CD40 activity, and use in the preparation of a medicament for treatment of a disease involving CD40 activity are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/686,596, filed Nov. 18, 2019, which claims the benefit of U.S.Provisional Application No. 62/769,514, filed Nov. 19, 2018, which ishereby incorporated in its entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 26, 2020, isnamed 200896_0015_01_US_604419.txt and is 167,708 bytes in size.

FIELD

The disclosure provides antibodies that bind CD40. The antibodypolypeptides bind CD40 and do not exhibit CD40 agonist activity. Theantibodies may comprise a modified IgG1 Fc domain, and exhibit minimalactivation of immature dendritic cells. Compositions comprisingantibodies, methods of use for treatment of diseases involving CD40activity, and use in the preparation of a medicament for treatment of adisease involving CD40 activity are provided.

BACKGROUND

CD40 is a co-stimulatory molecule belonging to the tumor necrosis factor(TNF) receptor superfamily that is present on antigen presenting cells(APC), including dendritic cells, B cells, and macrophages. APCs areactivated when CD40 binds its ligand, CD154 (CD40L), on T_(H) cells.CD40-mediated APC activation is involved in a variety of immuneresponses, including cytokine production, up-regulation ofco-stimulatory molecules (such as CD86), and enhanced antigenpresentation and B cell proliferation. CD40 can also be expressed byendothelial cells, smooth muscle cells, fibroblasts, and epithelialcells.

CD40 activation is also involved in a variety of undesired T cellresponses related to autoimmunity, transplant rejection, or allergicresponses, for example. One strategy for controlling undesirable T cellresponses is to target CD40 with an antagonistic antibody. For example,monoclonal antibody HCD122 (Lucatumumab), formerly known as Chiron 1212,is currently in clinical trials for the treatment of certainCD40-mediated inflammatory diseases. See “Study of HCD122 (Lucatumumab)and Bendamustine Combination Therapy in CD40⁺ Rituximab-RefractoryFollicular Lymphoma,” Clinical Trials Feeds, on the Internet athypertext transfer protocol:clinicaltrialsfeeds.org/clinical-trials/show/NCT01275209 (last updatedJan. 11, 2011). Monoclonal antibodies, however, can display agonistactivity. For example, the usefulness of the anti-CD40 antibody, Chi220,is limited by its weak stimulatory potential. See Adams, et al.,“Development of a chimeric anti-CD40 monoclonal antibody that synergizeswith LEA29Y to prolong islet allograft survival,” J. Immunol. 174:542-50 (2005).

SUMMARY

In a first embodiment, the present invention provides an isolatedantibody, or antigen binding portion thereof, that specifically binds tohuman CD40, wherein the antibody comprises a first polypeptide portioncomprising a heavy chain variable region, and a second polypeptideportion comprising a light chain variable region, wherein:

the heavy chain variable region comprises one of (i) a CDR1 comprisingSYWMH (SEQ ID NO: 1), a CDR2 comprising QINPTTGRSQYNEKFKT (SEQ ID NO:2), a CDR3 comprising WGLQPFAY (SEQ ID NO: 3); and (ii) a CDR1comprising SYWMH (SEQ ID NO: 1), a CDR2 comprising QINPSQGRSQYNEKFKT(SEQ ID NO: 12), a CDR3 comprising WGLQPFAY (SEQ ID NO: 3); and

the light chain variable region comprises a CDR1 comprising KASQDVSTAVA(SEQ ID NO: 7), a CDR2 comprising SASYRYT (SEQ ID NO: 8), and a CDR3comprising QQHYSTPWT (SEQ ID NO: 9).

The present invention further provides an isolated antibody or antigenbinding portion thereof, that specifically binds to human CD40, whereinthe antibody comprises a first polypeptide portion comprising a heavychain variable region, and a second polypeptide portion comprising alight chain variable region, wherein:

the heavy chain variable region comprises one of (i) a CDR1 consistingof SYWMH (SEQ ID NO: 1), a CDR2 consisting of QINPTTGRSQYNEKFKT (SEQ IDNO: 2), a CDR3 consisting of WGLQPFAY (SEQ ID NO: 3); and (ii) a CDR1consisting of SYWMH (SEQ ID NO: 1), a CDR2 consisting ofQINPSQGRSQYNEKFKT (SEQ ID NO: 12), a CDR3 consisting of WGLQPFAY (SEQ IDNO: 3); and

the light chain variable region comprises a CDR1 consisting ofKASQDVSTAVA (SEQ ID NO: 7), a CDR2 consisting of SASYRYT (SEQ ID NO: 8),and a CDR3 consisting of QQHYSTPWT (SEQ ID NO: 9).

The present invention further provides an isolated antibody or antigenbinding portion thereof, that specifically binds to human CD40, whereinthe antibody comprises a first polypeptide portion comprising a heavychain variable region, and a second polypeptide portion comprising alight chain variable region, wherein:

the heavy chain variable region comprises a CDR1 consisting of SYWMH(SEQ ID NO: 1), a CDR2 consisting QINPTTGRSQYNEKFKT (SEQ ID NO: 2), aCDR3 consisting of WGLQPFAY (SEQ ID NO: 3); and

the light chain variable region comprises a CDR1 consisting ofKASQDVSTAVA (SEQ ID NO: 7), a CDR2 consisting of SASYRYT (SEQ ID NO: 8),and a CDR3 consisting of QQHYSTPWT (SEQ ID NO: 9).

The present invention further provides an isolated antibody or antigenbinding portion thereof, that specifically binds to human CD40, whereinthe antibody comprises a first polypeptide portion comprising a heavychain variable region, and a second polypeptide portion comprising alight chain variable region, wherein:

the heavy chain variable region comprises the amino acid sequence of

(SEQ ID NO: 4) QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMG QINPTTGRS Q YNEKFKT RVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGL Q PFAYWGQGTLVTVSS,

and the light chain variable region comprises the amino acid sequence of

(SEQ ID NO: 10) DIQMTQSPSFLSASVGDRVTITC KASQDVSTAVA WYQQKPGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYSTPWT F GGGTKVEIK.

The present invention further provides an isolated antibody or antigenbinding portion thereof, that specifically binds to human CD40, whereinthe antibody comprises a first polypeptide portion comprising a heavychain variable region, and a second polypeptide portion comprising alight chain variable region, wherein the heavy chain variable regioncomprises the amino acid sequence of

(SEQ ID NO: 13) QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMGQINPS Q GRS Q YNEKFKT RVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGL Q PFAYWGQGTLVTVSS,and the light chain variable region comprises the amino acid sequence of

(SEQ ID NO: 16) EIVMTQSPATLSVSPGERATLSC KAS Q DVSTAVA WYQQKPGQAPRLLIYSASYRYT GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQ HYSTPWT F GGGTKVEIK.

The present invention further provides an isolated antibody or antigenbinding portion thereof, that specifically binds to human CD40, whereinthe antibody comprises a first polypeptide portion comprising a heavychain variable region, and a second polypeptide portion comprising alight chain variable region, wherein the heavy chain variable regioncomprises the amino acid sequence of

(SEQ ID NO: 4) QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMGQINPTTGRSQYNEKFKT RVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS,and the light chain variable region comprises the amino acid sequence of

(SEQ ID NO: 16) EIVMTQSPATLSVSPGERATLSC KASQDVSTAVA WYQQKPGQAPRLLIYSASYRYT GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQHYSTPWT F GGGTKVEIK.

In certain embodiments, the isolated antibody or antigen binding portionthereof comprises the first polypeptide portion comprising a human heavychain constant region; and the second polypeptide portion comprising ahuman light chain constant region. The isolated antibody or antigenbinding portion thereof described herein can comprise a human IgG1 Fcdomain comprising either (1) a mutation at Kabat position 238 thatreduces binding to Fc-gamma-receptors (FcγRs), wherein proline 238(P238) is mutated to one of the residues selected from the groupconsisting of lysine, serine, alanine, arginine, and tryptophan, andwherein the antibody or antigen binding portion thereof has reduced FcγRbinding; or (2) an alanine substituted at Kabat position 297.

The isolated antibody or antigen binding portion thereof describedherein can comprise a human IgG1 Fc domain comprising a mutation atKabat position 238 that reduces binding to Fc-gamma-receptors (FcγRs),wherein proline 238 (P238) is mutated to one of the residues selectedfrom the group consisting of lysine, serine, alanine, arginine, andtryptophan, and wherein the antibody or antigen binding portion hasreduced FcγR binding. In certain embodiments, P238 is mutated to lysine.

The isolated antibody or antigen binding portion thereof describedherein can comprise an Fc domain which comprises an amino acid sequenceselected from:

(SEQ ID NO: 22; IgG1-P238K (-C-term Lys)) EPKSCDKTHTCPPCPAPELLGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, (SEQ ID NO: 23; IgG1-P238K)EPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,(SEQ ID NO: 24; CH1-IgG1-P238K (-C-term Lys))ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGK SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, (SEQ ID NO: 25; CH1-IgG1-P238K)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV EPKSCDKTHTCPPCPAPELLGGK SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,(SEQ ID NO: 26; IgG1f-P238K (-C-term Lys))EPKSCDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, (SEQ ID NO: 27; IgG1f-P238K)EPKSCDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,(SEQ ID NO: 28; CH1-IgG1f-P238K (-C-term Lys))ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, or (SEQ ID No: 29; CH1-IgG1f-P238K)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In certain embodiments, the isolated antibody or antigen binding portionthereof described herein comprises a human IgG1 Fc domain comprisingeither (1) a mutation at Kabat position 238 that reduces binding toFc-gamma-receptors (FcγRs), wherein proline 238 (P238) is mutated to oneof the residues selected from the group consisting of lysine, serine,alanine, arginine, and tryptophan, and wherein the antibody or antigenbinding portion thereof has reduced FcγR binding; or (2) an alaninesubstituted at Kabat position 297, comprises a heavy chain variableregion comprising a CDR1 comprising SYWMH (SEQ ID NO: 1), a CDR2comprising QINPTTGRSQYNEKFKT (SEQ ID NO: 2), a CDR3 comprising WGLQPFAY(SEQ ID NO: 3); and a light chain variable region comprising a CDR1comprising KASQDVSTAVA (SEQ ID NO: 7), a CDR2 comprising SASYRYT (SEQ IDNO: 8), and a CDR3 comprising QQHYSTPWT (SEQ ID NO: 9).

The isolated antibody or antigen binding portion thereof can comprise ahuman IgG1 Fc domain comprising the amino acid sequence of SEQ ID NO: 22or SEQ ID NO: 23.

In some embodiments of the isolated antibody or antigen binding portionthereof described herein, the first polypeptide portion comprises orconsists of an amino acid sequence selected from the group consistingof:

(SEQ ID NO: 5; HC_Y12XX-hz28-CH1-IgG1-P238K- no terminal lysine)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMG QINPTTGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGLQPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG,(SEQ ID NO: 6; HC_Y12XX-hz28-CH1-IgG1-P238K-  with terminal lysine)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMG QINPTTGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGLQPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK,(SEQ ID NO: 30; HC_Y12XX-hz28-CH1-IgG1f-P238K-  no terminal lysine)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMG QINPTTGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGLQPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG, and(SEQ ID NO: 31; HC_Y12XX-hz28-CH1-IgG1f-P238K-  with terminal lysine)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMG QINPTTGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGLQPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGKSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK;and

the second polypeptide portion comprises or consists of the amino acidsequence of

(SEQ ID NO: 11; LC_Y12XX-hz28-CL) DIQMTQSPSFLSASVGDRVTITC KAS Q DVSTAVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYSTPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In some embodiments of the isolated antibody or antigen binding portionthereof described herein, the first polypeptide portion comprises orconsists of an amino acid sequence of

(SEQ ID NO: 5; HC_Y12XX-hz28-CH1-IgG1-P238K-  no terminal lysine)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQAPGQGLEWMG QINPTTGRS Q YNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAR WGL Q PFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG;and

the second polypeptide portion comprises or consists of the amino acidsequence of

(SEQ ID NO: 11; LC_Y12XX-hz28-CL) DIQMTQSPSFLSASVGDRVTITC KASQDVSTAVAWYQQKPGKAPKLLIY SA SYRYT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHYSTPWT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In certain embodiments, the isolated antibody or antigen binding portionthereof described herein comprises a human IgG1 Fc domain comprises ahuman IgG1 Fc domain comprising an alanine substituted at Kabat position297.

The isolated antibody or antigen binding portion thereof as describedherein can antagonize activities of CD40. The isolated antibody orantigen binding portion thereof described herein can be a chimericantibody. The isolated antibody or antigen binding portion thereofdescribed herein can be a humanized antibody. The isolated antibody orantigen binding portion thereof described herein can comprise a humanheavy chain constant region and a human light chain constant region.

The antibody or antigen binding portion thereof disclosed herein, is anantigen binding portion selected from the group consisting of Fv, Fab,F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, diabodies, and scFv-Fc. The isolatedantibody or antigen binding portion thereof as described herein is anscFv-Fc.

The antibody or antigen binding portion thereof disclosed herein canlinked to a therapeutic agent.

The antibody or antigen binding portion thereof disclosed herein can belinked to a second functional moiety having a different bindingspecificity than said antibody or antigen binding portion thereof.

The antibody or antigen binding portion thereof disclosed herein canfurther comprise an additional moiety.

A nucleic acid molecule encoding an isolated antibody or antigen bindingportion thereof is disclosed herein. An expression vector comprising thenucleic acid molecule is disclosed herein. Also contemplated is a celltransformed with the expression vector. Also disclosed is a method ofpreparing an anti-human CD40 antibody, or antigen binding portionthereof, comprising:

a) expressing the antibody, or antigen binding portion thereof, in thecell transformed with the expression vector comprising the nucleic acidmolecule encoding an isolated antibody or antigen binding portionthereof disclosed herein; and

b) isolating the antibody, or antigen binding portion thereof, from thecell.

Also provided is a pharmaceutical composition comprising: a) theantibody, or antigen binding portion thereof disclosed herein; and b) apharmaceutically acceptable carrier.

A method is provided of treating or preventing an immune response in asubject comprising administering to the subject the antibody, or theantigen binding portion thereof, disclosed herein. Further provided is amethod of treating or preventing an autoimmune or inflammatory diseasein a subject, comprising administering to the subject the antibody, orthe antigen binding portion, disclosed herein. Optionally, the antibody,or the antigen binding portion thereof, is administered with animmunosuppressive/immunomodulatory and/or anti-inflammatory agent.Administration may be simultaneous or sequential. An exemplary agent isa CTLA4 mutant molecule, such as L104EA29Y-Ig (belatacept). In suchmethod of treating or preventing an immune response in the subject, andin such method of treating or preventing an autoimmune or inflammatorydisease in a subject, preferably the subject has a disease selected fromthe group consisting of: Addison's disease, allergies, anaphylaxis,ankylosing spondylitis, asthma, atherosclerosis, atopic allergy,autoimmune diseases of the ear, autoimmune diseases of the eye,autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronaryheart disease, Crohn's disease, diabetes, epididymitis,glomerulonephritis, Graves' disease, Guillain-Barre syndrome,Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenicpurpura, inflammatory bowel disease, immune response to recombinant drugproducts (e.g., Factor VII in hemophiliacs), lupus nephritis, lupusnephritis, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.

Also contemplated is an antibody, or antigen binding portion thereof asdisclosed here, for use as a medicament. Further contemplated is anantibody, or antigen binding portion thereof as disclosed here, or amedicament comprising the same, for use to treat a subject in needthereof. Further contemplated is an antibody, or antigen binding portionthereof as disclosed herein in a therapeutically-effective amount, foruse in treating or preventing an immune response, wherein the antibodyor antigen binding portion thereof is for administering to a patient inneed thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D depict SPR sensorgram data of binding of antibodies to humanFcγRs. FIG. 1A depicts data for a control antibody, control IgG1. FIG.1B depicts data for Y12XX-hx28-IgG1-P238K, control IgG1. FIG. 1C depictsdata for a control antibody, Antibody B. FIG. 1D is a table summarizingthe KD values for Antibody/FcγRs interactions. The KD values wereobtained from either a 1:1 Langmuir fit (hCD64) or 1:1 steady state fit(hCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, and hCD16a-F158).

FIGS. 2A-2C depict iDC activation data for treatment of iDCs withhumanized Y12XX antibodies or control antibodies with or without theaddition of CD32a-expressing CHO cells. Increases in IL-6(interleukin-6) from the cell culture media and cell surface markerexpression, as indicated by flow cytometry mean fluorescence stainingwith anti-CD86 and anti-CD54 antibodies, were assessed. FIG. 2A depictsIL-6 data. FIG. 2B depicts CD86 data. FIG. 2C depicts CD54 data. Themean fluorescence intensity (MFI) is measured on the Y-axis in both FIG.2B and FIG. 2C. Each symbol represents data for iDC from an individualdonor. Y12XX-hz42-P238K was tested in cells from 4 donors,Y12XX-hz40-P238K was tested in cells from 6 donors, and Y12XX-hz28-P238Kwas tested in 10 donors. Concentration of antibody in is indicated (10,30, or 100 μg/ml). Inclusion of CHO-CD32 cells in the assay is asindicated to mediate FcγR mediated cross-linking or clustering. Ly6-IgGwas used as a negative control. Partial CD40 agonist 2141 andBMS986090-100 were used as positive controls.

FIGS. 3A and 3B depict exemplary data from complement dependentcytotoxicity (CDC) analysis of humanized Y12XX antibodies, CD40antibodies, and control antibodies. The CDC assay was performed twice.In the second assay, freshly thawed human complement serum was used.FIG. 3A depicts the data from the first iteration of the assay, and FIG.3B depicts the data from the second iteration of the assay.

FIGS. 4A and 4B depict exemplary data from antibody dependent cellularphagocytosis (ADCP) analysis of humanized Y12XX antibodies or controlantibodies, using CD14+ monocytes from two different donors as effectorcells. FIG. 4A depicts data obtained with donor #8 CD14+ monocytes aseffector cells. FIG. 4B depicts data obtained with donor #65 CD14+monocytes as effector cells.

FIGS. 5A and 5B depict exemplary data from antibody dependent cellularcytotoxicity (ADCC) analysis of humanized Y12XX antibodies or controlantibodies, using NK cells from two different donors as effector cells.FIG. 5A depicts data obtained with donor #38 NK cells as effector cells.FIG. 5B depicts data obtained with donor #55 NK cells as effector cells.

FIGS. 6A, 6B, 6C, and 6D depict data from an assay designed to assessthe NF-kB/AP-1 inducible SEAP activity on Ramos Blues Cells uponstimulation with different anti-CD40 antibodies Representative resultsfrom three independent studies for the activity of CD40 mAbs are shownin FIGS. 6A and 6B. FIGS. 6C and 6D depict data for the positivecontrol, CD40L-IZ. AIMV: AIM V™ medium (1×) (Thermo Fisher Scientific,Waltham, Mass.).

DETAILED DESCRIPTION

The present disclosure is directed to anti-CD40 antibodies, and inparticular, antagonistic anti-CD40 antibodies. For therapeutic targetssuch as CD40, FcγR-mediated cross-linking of anti-CD40 antibodies hasthe potential to lead to undesirable agonist signaling and potential fortoxicity. The present disclosure also describes antagonistic anti-CD40antibodies having reduced engagement of the “low affinity” FcγRs:hCD32a/FcγRIIa, hCD32b/FcγRIIb, and hCD16a/FcγRIIIa, as well as reducedengagement to “high affinity” FcγR hCD64. Reduced engagement of lowaffinity FcγRs is expected to reduce the likelihood of undesirableagonist signaling and undesirable potential for toxicity.

Definitions & Abbreviations

Further abbreviations and definitions are provided below.

-   APC antigen presenting cells-   CD54 also referred to as ICAM-1-   CDR complementarity determining regions-   C_(H) or CH constant heavy chain-   C_(L) or CL constant light chain-   CHO cell Chinese hamster ovary cell-   dAb domain antibody-   DC dendritic cell-   FcgR interchangeable with FcγR-   FcγR Fc-gamma-receptor-   FR Framework region-   GM-CSF granulocyte macrophage colony stimulating factor-   HC heavy chain-   ICAM-1 intracellular adhesion molecule 1-   iDC immature dendritic cells-   IFN interferon-   IgG immunoglobulin G-   IL-6 interleukin-6-   LC light chain-   mAb monoclonal antibody-   mg milligram-   ml or mL milliliter-   ng nanogram-   nM nanomolar-   pI isoelectric point-   SPR surface plasmon resonance-   TNF tumor necrosis factor-   μg microgram-   μM micromolar-   V_(L) or VL variable light chain domain-   Vk or VK kappa variable light chain domain-   V_(H) or VH variable heavy chain domain

In accordance with this detailed description, the followingabbreviations and definitions apply. It must be noted that as usedherein, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an antibody” includes a plurality of such antibodies andreference to “the dosage” includes reference to one or more dosages andequivalents thereof known to those skilled in the art, and so forth.

As used here, the term “about” is understood by persons of ordinaryskill in the art and will vary to some extent on the context in which itis used. Generally, “about” encompasses a range of values that areplus/minus 10% of a referenced value unless indicated otherwise in thespecification.

It is understood that any and all whole or partial integers between theranges set forth are included herein.

CD40 is also known and referred to as B-cell surface antigen CD40, Bp50,CD40L receptor, CDw40, CDW40, MGC9013, p50, TNFRSF5, and Tumor necrosisfactor receptor superfamily member 5. “Human CD40” refers to the CD40comprising the following amino acid sequence:

(SEQ ID NO: 20) MVRLPLQCVL WGCLLTAVHP EPPTACREKQ YLINSQCCSLCQPGQKLVSD CTEFTETECL PCGESEFLDT WNRETHCHQHKYCDPNLGLR VQQKGTSETD TICTCEEGWH CTSEACESCVLHRSCSPGFG VKQIATGVSD TICEPCPVGF FSNVSSAFEKCHPWTSCETK DLVVQQAGTN KTDVVCGPQD RLRALVVIPIIFGILFAILL VLVFIKKVAK KPTNKAPHPK QEPQEINFPDDLPGSNTAAP VQETLHGCQP VTQEDGKESR ISVQERQ.

As used herein, the term “variable domain” refers to immunoglobulinvariable domains defined by Kabat et al., Sequences of ImmunologicalInterest, 5th ed., U.S. Dept. Health & Human Services, Washington, D.C.(1991). The numbering and positioning of CDR amino acid residues withinthe variable domains is in accordance with the well-known Kabatnumbering convention. VH, “variable heavy chain” and “variable heavychain domain” refer to the variable domain of a heavy chain. VL,“variable light chain” and “variable light chain domain” refer to thevariable domain of a light chain.

The term “human,” when applied to antibodies, means that the antibodyhas a sequence, e.g., FR and/or CH domains, derived from a humanimmunoglobulin. A sequence is “derived from” a human immunoglobulincoding sequence when the sequence is either: (a) isolated from a humanindividual or from a cell or cell line from a human individual; (b)isolated from a library of cloned human antibody gene sequences or ofhuman antibody variable domain sequences; or (c) diversified by mutationand selection from one or more of the polypeptides above.

An “isolated” compound as used herein means that the compound is removedfrom at least one component with which the compound is naturallyassociated with in nature.

The anti-CD40 antibody of the present disclosure comprise a variableheavy chain and a variable light chain, each of which contains threecomplementarity-determining regions (CDRs) and four framework regions(FRs), arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs contain most ofthe residues that form specific interactions with the antigen and areprimarily responsible for antigen recognition.

The anti-CD40 antibody of the present disclosure can comprise CDRs ofhumanized antibody Y12XX-hz28 (Vh-hz14;Vk-hz2), Y12XX-hz40(Vh-hz12;Vk-hz3), or Y12XX-hz42 (Vh-hz14;Vk-hz3). An overview of theamino acid sequences of the heavy chain variable region and light chainvariable region is provided in Table 1. The table includes a short handname and a more detailed name for each amino acid sequence, as well asthe sequence identifiers.

TABLE 1 Antibody HC Variable Region LC Variable Region Y12XX-hz28Vh-hz14 Vk-hz2 (Y1268_IGHV1.6908- (Y1258_IGKV1.3902-Vk) S54T-N55T-Vh)(SEQ ID NO: 10) (SEQ ID NO: 4) Y12XX-hz40 Vy-hz12 Vk-hz3(Y1268_IGHV1.6908- (Y1258_IGKV3.1501-Vk) N55Q-Vh) (SEQ ID NO: 16) (SEQID NO: 13) Y12XX-hz42 Vh-hz14 Vk-hz3 (Y1268_IGHV1.6908-(Y1258_IGKV3.1501-Vk) S54T-N55T-Vh) (SEQ ID NO: 16) (SEQ ID NO: 4)

In a specific embodiment, the anti-CD40 antibodies of the presentdisclosure comprises the CDRs of humanized antibody Y12XX-hz28(Vh-hz14;Vk-hz2). Detail of the amino acid sequences of Y12XX-hz28 isprovided in Table 2.

TABLE 2 Y12XX-hz28 sequences (Vh-hz14; Vk-hz2) Heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ Vh-hz14 variable APGQGLEWMGQINPTTGRSQYNEKFKT RVTITADKSTST (SEQ ID NO: 4; CDRs regionAYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS underlined) SEQ ID NO: 4)VH-CDR1 SYWMH Amino acids 31-35 of SEQ ID NO: 1) SEQ ID NO: 4 VH-CDR2QINPTTGRSQYNEKFKT Amino acids 50-66 of (SEQ ID NO: 2) SEQ ID NO: 4VH-CDR3 WGLQPFAY Amino acids 99-106 of (SEQ ID NO: 3) SEQ ID NO: 4HC_Y12XX- QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ CDRs underlined:hz28-CH1- APGQGLEWMG QINPTTGRSQYNEKFKT RVTITADKSTST CH1 = amino acidsIgG1-P238K AYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS118-215 (italicized); (is IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVIgG1-P238K = amino acids with andSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT 216-446; P238K withoutQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL underlined; no C-terminal LGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV C-terminal lysine lysine)KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG (SEQ ID NO: 5)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ CDRs underlined; APGQGLEWMGQINPTTGRSQYNEKFKT RVTITADKSTST CH1 = amino acids AYMELSSLRSEDTAVYYCARWGLQPFAY WGQGTLVTVSS 118-215 (italicized);ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV IgG1-P238K = amino acidsSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT 216-447; P238KQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL underlined; C-terminal LGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV lysine presentKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK(SEQ ID NO: 6) Light chain DIQMTQSPSFLSASVGDRVTITC KASQDVSTAVA WYQQKVk-hz2 variable PGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTLTISSL(SEQ ID NO: 10; CDRs region QPEDFATYYC QQHYSTPWT FGGGTKVEIK underlined)(SEQ ID NO: 10) VL-CDR1 KASQDVSTAVA Amino acids 24-34 of (SEQ ID NO: 7)SEQ ID NO: 10 VL-CDR2 SASYRYT Amino acids 50-56 of (SEQ ID NO: 8)SEQ ID NO: 10 VL-CDR3 QQHYSTPWT Amino acids 89-97 of (SEQ ID NO: 9)SEQ ID NO: 10 LC_Y12XX- DIQMTQSPSFLSASVGDRVTITC KASQDVSTAVA WYQQKCDRs underlined; hz28 PGKAPKLLIY SASYRYT GVPSRFSGSGSGTDFTLTISSLCL = amino acids 108-214 QPEDFATYYC QQHYSTPWT FGGGTKVEIKRTVAAPSVFI(italicized) FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC(SEQ ID NO: 11)

In a specific embodiment, the anti-CD40 antibody of the presentdisclosure comprises the CDRs of humanized antibody Y12XX-hz40(Vh-hz12;Vk-hz3). Amino acid sequences of Y12XX-hz40 are provided inTable 3.

TABLE 3 Y12XX-hz40 sequences (Vh-hz12; Vk-hz3) Heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ Vh-hz12 (SEQ ID NO: 13;variable APGQGLEWMG QINPSQGRSQYNEKFKT RVTITADKSTST CDRs underlined)region AYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS (SEQ ID NO: 13) VH-CDR1SYWMH Amino acids 31-35 of (SEQ ID NO: 1) SEQ ID NO: 13 VH-CDR2QINPSQGRSQYNEKFKT Amino acids 50-66 of (SEQ ID NO: 12) SEQ ID NO: 13VH-CDR3 WGLQPFAY Amino acids 99-106 of (SEQ ID NO: 3) SEQ ID NO: 13HC_Y12XX- QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ CDRs underlined;hz40-P238K- APGQGLEWMG QINPSQGRSQYNEKFKT RVTITADKSTST CH1 = amino acidsIgG1a with AYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS118-215 (italicized); and withoutASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV IgG1-P238K = amino acidsC-terminal SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT216-446; P238K underlined; lysineQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL no C-terminal lysine LGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG(SEQ ID NO: 14) QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQCDRs underlined; APGQGLEWMG QINPSQGRSQYNEKFKT RVTITADKSTSTCH1 = amino acids AYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS118-215 (italicized); ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVIgG1-P238K = amino acids SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT216-447; P238K underlined; QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELC-terminal lysine present LGG K SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK(SEQ ID NO: 15) Light chain EIVMTQSPATLSVSPGERATLSC KASQDVSTAVA WYQQKVk-hz3 (SEQ ID NO: 16; variable PGQAPRLLIY SASYRYTGIPARFSGSGSGTEFTLTISSL CDRs underlined) region QSEDFAVYYC QQHYSTPWTFGGGTKVEIK (SEQ ID NO: 16) VL-CDR1 KASQDVSTAVA Amino acids 24-34 of SEQ (SEQ ID NO: 7) ID NO: 16 VL-CDR2 SASYRYT Amino acids 50-56 of SEQ (SEQ ID NO: 8) ID NO: 16 VL-CDR3 QQHYSTPWT Amino acids 89-97 of SEQ (SEQ ID NO: 9) ID NO: 16 LC_Y12XX- EIVMTQSPATLSVSPGERATLSC KASQDVSTAVAWYQQK CDRs underlined; hz40 PGQAPRLLIY SASYRYT GIPARFSGSGSGTEFTLTISSLCL = amino acids 108-214 QSEDFAVYYC QQHYSTPWT FGGGTKVEIKRTVAAPSVFI(italicized) FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC(SEQ ID NO: 17)

In a specific embodiment, the anti-CD40 antibody of the presentdisclosure comprises the CDRs of humanized antibody Y12XX-hz42(Vh-hz14;Vk-hz3). Detail of the amino acid sequences of Y12XX-hz42 isprovided in Table 4.

TABLE 4 Y12XX-hz42 sequences (Vh-hz14; Vk-hz3) Heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ Vh-hz14 variable APGQGLEWMGQINPTTGRSQYNEKFKT RVTITADKSTST (SEQ ID NO: 4; CDRs regionAYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS underlined) SEQ ID NO: 4)VH-CDR1 SYWMH Amino acids 31-35 of SEQ ID NO: 1) SEQ ID NO: 4 VH-CDR2QINPTTGRSQYNEKFKT Amino acids 50-66 of (SEQ ID NO: 2) SEQ ID NO: 4VH-CDR3 WGLQPFAY Amino acids 99-106 of (SEQ ID NO: 3) SEQ ID NO: 4HC_Y12XX- QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ CDRs underlined;hz42-P238K- APGQGLEWMG QINPTTGRSQYNEKFKT RVTITADKSTST CH1 = amino acidsIgG1a with AYMELSSLRSEDTAVYYCAR WGLQPFAY WGQGTLVTVSS118-215 (italicized); and withoutASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV IgG1-P238K = amino acidsC-terminal SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT 216-446; P238K lysineQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL underlined; no LGG KSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV C-terminal lysineKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG (SEQ ID NO: 5)QVQLVQSGAEVKKPGSSVKVSCKASGYAFT SYWMH WVRQ CDRs underlined; APGQGLEWMGQINPTTGRSQYNEKFKT RVTITADKSTST CH1 = amino acids AYMELSSLRSEDTAVYYCARWGLQPFAY WGQGTLVTVSS 118-215 (italicized);ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV IgG1-P238K = amino acidsSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT 216-447; P238KQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEL underlined; C-terminal LGG KSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEV lysine presentKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK(SEQ ID NO: 6) Light chain EIVMTQSPATLSVSPGERATLSC KASQDVSTAVA WYQQKVk-hz3 (SEQ ID NO: 16; variable PGQAPRLLIY SASYRYTGIPARFSGSGSGTEFTLTISSL CDRs underlined) region QSEDFAVYYC QQHYSTPWTFGGGTKVEIK (SEQ ID NO: 16) VL-CDR1 KASQDVSTAVA Amino acids 24-34 of(SEQ ID NO: 7) SEQ ID NO: 16 VL-CDR2 SASYRYT Amino acids 50-56 of(SEQ ID NO: 8) SEQ ID NO: 16 VL-CDR3 QQHYSTPWT Amino acids 89-97 of(SEQ ID NO: 9) SEQ ID NO: 16 LC_ Y12XX- EIVMTQSPATLSVSPGERATLSCKASQDVSTAVA WYQQK CDRs underlined; hz42 PGQAPRLLIY SASYRYTGIPARFSGSGSGTEFTLTISSL CL = amino acids 108-214 QSEDFAVYYC QQHYSTPWTFGGGTKVEIKRTVAAPSVFI (italicized)FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC(SEQ ID NO: 17)

In one embodiment, the antibodies of the disclosure can comprise theamino acid sequences of the CDR1, CDR2, and CDR3 regions of thehumanized Y12XX-hz28 variable heavy and light chains sequences (seee.g., SEQ ID NOS: 4 and 10 respectively, as an example). Monoclonalantibodies contain all 6 CDRs (3 for the V_(H) and 3 for the V_(L)), forexample, SYWMH (SEQ ID NO: 1), QINPTTGRSQYNEKFKT (SEQ ID NO: 2), andWGLQPFAY (SEQ ID NO: 3) for the variable heavy chain CDRs 1-3respectively and KASQDVSTAVA (SEQ ID NO: 7), SASYRYT (SEQ ID NO: 8), andQQHYSTPWT (SEQ ID NO: 9) for the variable light chain CDRs 1-3respectively.

In one embodiment, the antibodies of the disclosure can comprise theamino acid sequences of the CDR1, CDR2, and CDR3 regions of thehumanized Y12XX-hz40 variable heavy and light chains sequences (seee.g., SEQ ID NOS: 13 and 16 respectively, as an example). Monoclonalantibodies contain all 6 CDRs (3 for the V_(H) and 3 for the V_(L)), forexample, SYWMH (SEQ ID NO: 1), QINPSQGRSQYNEKFKT (SEQ ID NO: 12), andWGLQPFAY (SEQ ID NO: 3) for the variable heavy chain CDRs 1-3respectively and KASQDVSTAVA (SEQ ID NO: 7), SASYRYT (SEQ ID NO: 8), andQQHYSTPWT (SEQ ID NO: 9) for the variable light chain CDRs 1-3respectively.

In one embodiment, the antibodies of the disclosure can comprise theamino acid sequences of the CDR1, CDR2, and CDR3 regions of thehumanized Y12XX-hz42 variable heavy and light chains sequences (seee.g., SEQ ID NOS: 4 and 16 respectively, as an example). Monoclonalantibodies contain all 6 CDRs (3 for the V_(H) and 3 for the V_(L)), forexample, SYWMH (SEQ ID NO: 1), QINPTTGRSQYNEKFKT (SEQ ID NO: 2), andWGLQPFAY (SEQ ID NO: 3) for the variable heavy chain CDRs 1-3respectively and KASQDVSTAVA (SEQ ID NO: 7), SASYRYT (SEQ ID NO: 8), andQQHYSTPWT (SEQ ID NO: 9) for the variable light chain CDRs 1-3respectively.

An “antibody” (Ab) shall include, without limitation, an immunoglobulinwhich binds specifically to an antigen and comprises at least two heavy(H) chains and two light (L) chains interconnected by disulfide bonds,or an antigen-binding portion thereof. Each H chain comprises a heavychain variable region (abbreviated herein as V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threeconstant domains, C_(H1), C_(H2) and C_(H3). Each light chain comprisesa light chain variable region (abbreviated herein as V_(L)) and a lightchain constant region. The light chain constant region comprises oneconstant domain, C_(L). The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDRs), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) comprisesthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen.

An “antigen binding portion” of an Ab (also called an “antigen-bindingfragment”) or antigen binding portion thereof refers to one or moresequences of an Ab (full length or fragment of the full length antibody)that retain the ability to bind specifically to the antigen bound by thewhole Ab. Examples of an antigen-binding fragment include Fab, F(ab′)₂,scFv (single-chain variable fragment), Fab′, dsFv, sc(Fv)2, and scFv-Fc.

A “humanized” antibody refers to an Ab in which some, most or all of theamino acids outside the CDR domains of a non-human Ab are replaced withcorresponding amino acids derived from human immunoglobulins. In oneembodiment of a humanized form of an Ab, some, most or all of the aminoacids outside the CDR domains have been replaced with amino acids fromhuman immunoglobulins, whereas some, most or all amino acids within oneor more CDR regions are unchanged. Small additions, deletions,insertions, substitutions or modifications of amino acids arepermissible as long as they do not abrogate the ability of the Ab tobind to a particular antigen. A “humanized” Ab retains an antigenicspecificity similar to that of the original Ab.

A “chimeric antibody” refers to an Ab in which the variable regions arederived from one species and the constant regions are derived fromanother species, such as an Ab in which the variable regions are derivedfrom a mouse Ab and the constant regions are derived from a human Ab.

As used herein, “specific binding” refers to the binding of an antigenby an antibody with a dissociation constant (K_(d)) of about 1 μM orlower as measured, for example, by surface plasmon resonance (SPR).Suitable assay systems include the BIAcore™ (GE Healthcare LifeSciences, Marlborough, Mass.) surface plasmon resonance system andBIAcore™ kinetic evaluation software (e.g., version 2.1).

Binding of the present antibodies to CD40 antagonizes at least one CD40activity. “CD40 activities” include, but are not limited to, T cellactivation (e.g., induction of T cell proliferation or cytokinesecretion), macrophage activation (e.g., the induction of reactiveoxygen species and nitric oxide in the macrophage), and B cellactivation (e.g., B cell proliferation, antibody isotype switching, ordifferentiation to plasma cells). CD40 activities can be mediated byinteraction with other molecules. “CD40 activities” include thefunctional interaction between CD40 and the following molecules, whichare identified by their Uniprot Accession Number is parentheses:

CALR (P27797); ERP44 (Q9BS26); FBL (P22087); POLR2H (P52434); RFC5(P40937); SGK1 (O00141); SLC30A7 (Q8NEW0); SLC39A7 (Q92504); TRAF2(Q5T1L5); TRAF3 (Q13114); TRAF6 (Q9Y4K3); TXN (Q5T937); UGGT1 (Q9NYU2);and USP15 (Q9Y4E8).

For example, a CD40 “activity” includes an interaction with TRAF2.CD40/TRAF2 interaction activates NF-κB and JNK. See Davies et al., Mol.Cell Biol. 25: 9806-19 (2005). This CD40 activity thus can be determinedby CD40-dependent cellular NF-κB and JNK activation, relative to areference.

As used herein, the terms “activate,” “activates,” and “activated” referto an increase in a given measurable CD40 activity by at least 10%relative to a reference, for example, at least 10%, 25%, 50%, 75%, oreven 100%, or more. A CD40 activity is “antagonized” if the CD40activity is reduced by at least 10%, and in an exemplary embodiment, atleast about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, or even100% (i.e., no detectable activity), relative to the absence of theantagonist. For example, an antibody may antagonize some or all CD40activity, while not activating CD40. For example, the antibody may notactivate B cell proliferation. The antibody may not activate cytokinesecretion by T cells, where the cytokine is at least one cytokineselected from the group consisting of IL-2, IL-6, IL-10, IL-13, TNF-α,and IFN-γ.

Variable domains may comprise one or more framework regions (FR) withthe same amino acid sequence as a corresponding framework region encodedby a human germline antibody gene segment. Preferred framework sequencesfor use in the antibodies described herein are those that arestructurally similar to the framework sequences used by antibodiesdescribed herein. The V_(H) CDR1, 2 and 3 sequences, and the V_(L) CDR1,2 and 3 sequences, can be grafted onto framework regions that have theidentical sequence as that found in the germline immunoglobulin genefrom which the framework sequence derive, or the CDR sequences can begrafted onto framework regions that contain up to 20, preferablyconservative, amino acid substitutions as compared to the germlinesequences. For example, it has been found that in certain instances itis beneficial to mutate residues within the framework regions tomaintain or enhance the antigen binding ability of the antibody (seee.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.).

Exemplary framework regions include but are not limited to those inTables 5 and 6 below.

TABLE 5 Heavy Framework Region Sequence FR1 Amino acid residues 1-30 ofany VH sequence in Table 8 (SEQ ID NOs: 53- 75) or Table 10 (SEQ ID NOs:4, 13, and 99-113) in the Examples FR2 Amino acid residues 36-49 of anyVH sequence in Table 8 (SEQ ID NOs: 53- 75) or Table 10 (SEQ ID NOs: 4,13, and 99-113) in the Examples FR3 Amino acid residues 67-98 of any VHsequence in Table 8 (SEQ ID NOs: 53- 75) or Table 10 (SEQ ID NOs: 4, 13,and 99-113) in the Examples FR4 Amino acid residues 107-117 of any VHsequence in Table 8 (SEQ ID NOs: 53- 75) or Table 10 (SEQ ID NOs: 4, 13,and 99-113) in the Examples

TABLE 6 Light Framework Region Sequence FR1 Amino acid residues 1-23 ofany VL sequence in Table 8 (SEQ ID NOs: 76- 98) or Table 10 (SEQ ID NOs:10, 16, and 114-116) in the Examples FR2 Amino acid residues 35-49 ofany VL sequence in Table 8 (SEQ ID NOs: 76- 98) or Table 10 (SEQ ID NOs:10, 16, and 114-116) in the Examples FR3 Amino acid residues 57-88 ofany VL sequence in Table 8 (SEQ ID NOs: 76- 98) or Table 10 (SEQ ID NOs:10, 16, and 114-116) in the Examples FR4 Amino acid residues 98-107 ofany VL sequence in Table 8 (SEQ ID NOs: 76- 98) or Table 10 (SEQ ID NOs:10, 16, and 114-116) in the Examples

A variant variable domain may differ from the variable domain of thehumanized Y12XX-hz28, Y12XX-hz40, or Y12XX-hz42 sequence by up to 10amino acids or any integral value between, where the variant variabledomain specifically binds CD40. Alternatively, the variant variabledomain may have at least 90% sequence identity (e.g., at least 92%, 95%,98%, or 99% sequence identity) relative to the sequence of the humanizedY12XX-hz28, Y12XX-hz40, or Y12XX-hz42 sequence, respectively.Non-identical amino acid residues or amino acids that differ between twosequences may represent amino acid substitutions, additions, ordeletions. Residues that differ between two sequences appear asnon-identical positions, when the two sequences are aligned by anappropriate amino acid sequence alignment algorithm, such as BLAST® (aregistered trademark of the U.S. National Library of Medicine).

Exemplary CD40 antibodies of the present invention can include anisolated antibody, or antigen binding portion thereof, that specificallybinds to human CD40, wherein said antibody comprises a first polypeptideportion comprising a heavy chain variable region, and a secondpolypeptide portion comprising a light chain variable region, wherein:

said heavy chain variable region comprises one of (i) a CDR1 comprisingSYWMH (SEQ ID NO: 1), a CDR2 comprising QINPTTGRSQYNEKFKT (SEQ ID NO:2), a CDR3 comprising WGLQPFAY (SEQ ID NO: 3) and (ii) a CDR1 comprisingSYWMH (SEQ ID NO: 1), a CDR2 comprising QINPSQGRSQYNEKFKT (SEQ ID NO:12), a CDR3 comprising WGLQPFAY (SEQ ID NO: 3); and

said light chain variable region comprises a CDR1 comprising KASQDVSTAVA(SEQ ID NO: 7), a CDR2 comprising SASYRYT (SEQ ID NO: 8), and a CDR3comprising QQHYSTPWT (SEQ ID NO: 9).

The isolated antibody or antigen binding portion thereof can antagonizeone or more activities of CD40. The isolated antibody or antigen bindingportion thereof can be a chimeric antibody. Exemplary heavy and lightvariable chains for a chimeric antibody are in Table 8 of the Examples.The isolated antibody or antigen binding portion thereof can be ahumanized antibody. Exemplary humanized heavy and light variable chainsare in Table 10 of the Examples. The isolated antibody or antigenbinding portion thereof can comprise a human heavy chain constant regionand a human light chain constant region.

Fc Domain and Constant Region

The carboxyl-terminal “half” of a heavy chain defines a constant region(Fc) and which is primarily responsible for effector function. As usedherein, the term “Fc domain” refers to the constant region antibodysequences comprising CH2 and CH3 constant domains as delimited accordingto Kabat et al., Sequences of Immunological Interest, 5^(th) ed., U.S.Dept. Health & Human Services, Washington, D.C. (1991). The Fc regionmay be derived from a human IgG. For instance, the Fc region may bederived from a human IgG1 or a human IgG4 Fc region. A heavy variabledomain can be fused to an Fc domain. The carboxyl terminus of thevariable domain may be linked or fused to the amino terminus of the FcCH2 domain. Alternatively, the carboxyl terminus of the variable domainmay be linked or fused to the amino terminus of a linker amino acidsequence, which itself is fused to the amino terminus of an Fc domain.Alternatively, the carboxyl terminus of the variable domain may belinked or fused to the amino terminus of a CH1 domain, which itself isfused to the Fc CH2 domain. Optionally, the protein may comprise thehinge region after the CH1 domain in whole or in part. Optionally anamino acid linker sequence is present between the variable domain andthe Fc domain. The carboxyl terminus of the light variable domain may belinked or fused to the amino terminus of a CL domain.

An exemplary sequence for a heavy chain CH1 is amino acids 118-215 ofSEQ ID NO: 5 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV; SEQ ID NO: 18). An exemplarysequence for a light chain CL is amino acids 108-214 of SEQ ID NO: 11(RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; SEQ ID NO: 19).

The antibody can be a fusion antibody comprising a first variable domainthat specifically binds human CD40, and a second domain comprising an Fcdomain.

Exemplary Fc domains used in the fusion protein can include human IgGdomains. Exemplary human IgG Fc domains include IgG4 Fc domain and IgG1Fc domain. While human IgG heavy chain genes encode a C-terminal lysine,the lysine is often absent from endogenous antibodies as a result ofcleavage in blood circulation. Antibodies having IgG heavy chainsincluding a C-terminal lysine, when expressed in mammalian cellcultures, may also have variable levels of C-terminal lysine present(Cai et al, 2011, Biotechnol Bioeng. 108(2): 404-12). Accordingly, theC-terminal lysine of any IgG heavy chain Fc domain disclosed herein maybe omitted.

The isolated antibody or antigen binding portion thereof describedherein, can comprise an Fc domain which comprises an amino acid sequenceof:

(Fc consensus; SEQ ID NO: 21)EPKSCDKTHTCPPCPAPELLGG(P/K)SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY(N/A)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR(D/E)E(L/M)TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG(K/not present).The parenthetical notation indicates possible amino acid identities atthe position. For instance, Kabat position 238 can be either Proline (P)or Lysine (K), which is notated as (P/K). Additional exemplary,non-limiting consensus sequences are SEQ ID NOs: 118-120.

The isolated antibody or antigen binding portion thereof describedherein can comprise a human IgG1 Fc domain comprising a mutation atKabat position 238 that reduces binding to Fc-gamma-receptors (FcγRs),wherein proline 238 (P238) is mutated to one of the residues selectedfrom the group consisting of lysine (K), serine (S), alanine (A),arginine (R) and tryptophan (W), and wherein the antibody or antigenbinding portion thereof has reduced FcγR binding. The isolated antibodyor antigen binding portion thereof described herein can have P238mutated to lysine in a human IgG1 Fc domain.

The isolated antibody or antigen binding portion thereof comprises an Fcdomain which comprises an amino acid sequence selected from: SEQ ID NOs:22-29.

Exemplary sequences comprising the IgG1 Fc domains above include: SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 30, and SEQ ID NO: 31.

The isolated antibody or antigen binding portion thereof describedherein can comprise a human IgG1 Fc domain comprising an alaninesubstituted at Kabat position 297. For example, the isolated antibody orantigen binding portion thereof comprises an Fc domain which comprisesan amino acid sequence selected from: SEQ ID NOs: 32-39.

The isolated antibody or antigen binding portion described herein maycomprise (1) a variable heavy chain (V_(H)) selected from Table 8 orTable 10 in the Examples, or the CDRs thereof, and/or (2) a variablelight chain (V_(L)) selected from Table 8 or Table 10 in the Examples,or the CDRs thereof.

The isolated antibody or antigen binding portion thereof disclosedherein may comprise a heavy chain amino acid sequence selected fromVh-hz12 (SEQ ID NO: 13) and Vh-hz14 (SEQ ID NO: 4).

The isolated antibody or antigen binding portion thereof disclosedherein may comprise a light chain amino acid sequence selected fromVk-hz2 (SEQ ID NO: 10) and Vk-hz3 (SEQ ID NO: 16).

The isolated antibody or antigen binding portion thereof disclosedherein may be an antibody selected from the group consisting of:

a) Y12XX-hz28-P238K having a heavy chain of SEQ ID NO: 5 or 6 and lightchain of SEQ ID NO: 11;

b) Y12XX-hz40-P238K having a heavy chain of SEQ ID NO: 14 or 15 andlight chain of SEQ ID NO: 17; and

c) Y12XX-hz42-P238K having a heavy chain of SEQ ID NO: 5 or 6 and lightchain of SEQ ID NO: 17.

The antibody or antigen binding portion thereof disclosed herein,wherein the antigen binding portion is selected from the groupconsisting of Fv, Fab, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)₂, diabodies,and scFv-Fc.

The antibody or antigen binding portion thereof disclosed herein can bean immunoconjugate, wherein the antibody or antigen-binding portionthereof is linked to a therapeutic agent.

The antibody or antigen binding portion thereof disclosed herein can bea bispecific antibody, wherein the antibody or antigen-binding portionthereof is linked to a second functional moiety having a differentbinding specificity than said antibody or antigen binding portionthereof.

The antibody or antigen binding portion thereof disclosed herein canfurther comprise an additional moiety.

The variable regions of the present antibodies may optionally be linkedto the Fc domain by an “amino acid linker” or “linker.” For example, theC-terminus of a variable heavy chain domain may be fused to theN-terminus of an amino acid linker, and an Fc domain may be fused to theC-terminus of the linker. Although amino acid linkers can be any lengthand consist of any combination of amino acids, the linker length may berelatively short (e.g., five or fewer amino acids) to reduceinteractions between the linked domains. The amino acid composition ofthe linker also may be adjusted to reduce the number of amino acids withbulky side chains or amino acids likely to introduce secondarystructure. Suitable amino acid linkers include, but are not limited to,those up to 3, 4, 5, 6, 7, 10, 15, 20, or 25 amino acids in length.Representative amino acid linker sequences include GGGGS (SEQ ID NO:40), and a linker comprising 2, 3, 4, or 5 copies of GGGGS (SEQ ID NOs:41 to 44, respectively). TABLE 7 lists suitable linker sequences for usein the present disclosure.

TABLE 7 Representative Linker Sequences GGGGS SEQ ID NO: 40 (GGGGS)₂SEQ ID NO: 41 (GGGGS)₃ SEQ ID NO: 42 (GGGGS)4 SEQ ID NO: 43 (GGGGS)₅SEQ ID NO: 44 AST SEQ ID NO: 45 TVAAPS SEQ ID NO: 46 TVA SEQ ID NO: 47ASTSGPS SEQ ID NO: 48Antibody Preparation

The antibody can be produced and purified using ordinary skill in asuitable mammalian host cell line, such as CHO, 293, COS, NSO, and thelike, followed by purification using one or a combination of methods,including protein A affinity chromatography, ion exchange, reverse phasetechniques, or the like.

As well known in the art, multiple codons can encode the same aminoacid. Nucleic acids encoding a protein sequence thus include nucleicacids having codon degeneracy. The polypeptide sequences disclosedherein can be encoded by a variety of nucleic acids. The genetic code isuniversal and well known. Nucleic acids encoding any polypeptidesequence disclosed herein can be readily conceived based on conventionalknowledge in the art as well as optimized for production. While thepossible number of nucleic acid sequence encoding a given polypeptide islarge, given a standard table of the genetic code, and aided by acomputer, the ordinarily skilled artisan can easily generate everypossible combination of nucleic acid sequences that encode a givenpolypeptide.

A representative nucleic acid sequence encoding the Y12XX heavy chainvariable domain of Y12XX-hz28 including a constant region CH1 and Fcdomain IgG1-P238K is:

(SEQ ID NO: 49) ATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGAGAGCGCTCGCACAGGTGCAGCTGGTGCAGTCTGGTGCCGAGGTCAAAAAGCCAGGCTCCAGCGTGAAGGTGAGCTGCAAGGCCTCTGGCTACGCTTTCACCTCTTATTGGATGCACTGGGTGAGACAGGCTCCTGGACAGGGCCTGGAGTGGATGGGCCAGATCAACCCAACCACCGGCAGAAGCCAGTACAATGAGAAGTTTAAGACCCGCGTGACCATCACAGCCGACAAGTCCACCAGCACAGCTTATATGGAGCTGTCTTCCCTGAGGTCCGAGGATACAGCCGTGTACTATTGCGCTCGGTGGGGCCTGCAGCCTTTCGCTTACTGGGGCCAGGGCACCCTGGTGACAGTGAGCTCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCCGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGAAAGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTGA.In this sequence, nucleotides 1-51 encode a signal peptide (optional),nucleotides 52-402 encode the heavy chain variable region in whichnucleotides 141-155 encode CDR1, nucleotides 198-249 encode CDR2, andnucleotides 346-369 encode CDR3 of the Y12XX variable domain of theheavy chain. Nucleotides 403-696 encode a CH1 domain, and nucleotides697-1399 encode IgG1-P238K. Nucleotides 1400-1402 are a stop codon.

A representative nucleic acid sequence encoding the Y12XX light chainvariable domain of Y12XX-hz28 including a constant region CL is:

(SEQ ID NO: 50) ATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGCGCGCCTTGGCCGACATCCAGATGACCCAGTCCCCCTCCTTCCTGTCTGCCTCCGTGGGCGACAGAGTGACCATCACCTGTAAGGCTTCCCAGGATGTGAGCACAGCCGTGGCTTGGTACCAGCAGAAGCCAGGCAAGGCCCCCAAGCTGCTGATCTATTCCGCCTCTTACAGGTATACCGGCGTGCCCTCTCGGTTCTCCGGCAGCGGCTCTGGCACAGACTTTACCCTGACAATCTCCAGCCTGCAGCCTGAGGATTTCGCCACCTACTATTGCCAGCAGCACTACTCCACCCCATGGACATTTGGCGGCGGCACCAAGGTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG.In this sequence, nucleotides 1-51 encode a signal peptide (optional),nucleotides 52-372 encode the light chain variable region in whichnucleotides 121-153 encode CDR1, nucleotides 199-219 encode CDR2, andnucleotides 316-342 encode CDR3. Nucleotides 373-693 encode a CL.Nucleotides 694-696 are a stop codon

The coding sequence for the heavy and/or light chain optionally mayencode a signal peptide, such as MRAWIFFLLCLAGRALA (SEQ ID NO: 51), atthe 5′ end of the coding sequence. As described above, an exemplarynucleic acid coding sequence for this signal peptide isATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGAGAGCGCTCGCA (SEQ ID NO: 52).

Accordingly, a nucleic acid encoding an antibody disclosed herein isalso contemplated. Such a nucleic acid may be inserted into a vector,such as a suitable expression vector, e.g., pHEN-1 (Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133-4137). Further provided is anisolated host cell comprising the vector and/or the nucleic acid.

The antibody of the disclosure can be produced and purified using onlyordinary skill in any suitable mammalian host cell line, such as CHO(Chinese hamster ovary cells), 293 (human embryonic kidney 293 cells),COS cells, NSO cells, and the like, followed by purification using oneor a combination of methods, including protein A affinitychromatography, ion exchange, reverse phase techniques, or the like.

Pharmaceutical Compositions and Methods of Treatment

A pharmaceutical composition comprises a therapeutically-effectiveamount of one or more antibodies and optionally a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers include, forexample, water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like, as well as combinations thereof. Pharmaceuticallyacceptable carriers can further comprise minor amounts of auxiliarysubstances, such as wetting or emulsifying agents, preservatives, orbuffers that enhance the shelf-life or effectiveness of the fusionprotein. The compositions can be formulated to provide quick, sustained,or delayed release of the active ingredient(s) after administration.Suitable pharmaceutical compositions and processes for preparing themare known in the art. See, e.g., Remington, THE SCIENCE AND PRACTICE OFPHARMACY, A. Gennaro, et al., eds., 21st ed., Mack Publishing Co.(2005).

The pharmaceutical composition may be administered alone or incombination therapy, (i.e., simultaneously or sequentially) with animmunosuppressive/immunomodulatory and/or anti-inflammatory agent. Anexemplary type of agent is a cytotoxic T lymphocyte-associated protein 4(CTLA4) mutant molecule. An exemplary CTLA4 mutant molecule isL104EA29Y-Ig (belatacept) which is a modified CTLA4-Ig. Different immunediseases can require use of specific auxiliary compounds useful fortreating immune diseases, which can be determined on apatient-to-patient basis. For example, the pharmaceutical compositionmay be administered in combination with one or more suitable adjuvants,e.g., cytokines (IL-10 and IL-13, for example) or other immunestimulators, e.g., chemokines, tumor-associated antigens, and peptides.Suitable adjuvants are known in the art.

A method of treating an immune disease in a patient in need of suchtreatment may comprise administering to the patient a therapeuticallyeffective amount of the antibody, or antigen binding portion thereof, asdescribed herein. Further provided is a method of treating or preventingan autoimmune or inflammatory disease in a patient in need of suchtreatment may comprise administering to the patient a therapeuticallyeffective amount of the antibody, or antigen binding portion thereof, asdescribed herein. Also provided is the use of an antibody, or antigenbinding portion thereof, of the disclosure, or a pharmaceuticallyacceptable salt thereof, for treating an immune disease in a patient inneed of such treatment and/or for treating or preventing an autoimmuneor inflammatory disease in a patient in need of such treatment, that maycomprise administering to the patient a therapeutically effective amountof the antibody, or antigen binding portion thereof. AntagonizingCD40-mediated T cell activation could inhibit undesired T cell responsesoccurring during autoimmunity, transplant rejection, or allergicresponses, for example. Inhibiting CD40-mediated T cell activation couldmoderate the progression and/or severity of these diseases.

The use of an antibody, or antigen binding portion thereof, of thedisclosure, or a pharmaceutically acceptable salt thereof, in thepreparation of a medicament for treatment of an immune disease and/orfor treating or preventing an autoimmune or inflammatory disease in apatient in a patient in need of such treatment, is also provided. Themedicament can, for example, be administered in combination with animmunosuppressive/immunomodulatory and/or anti-inflammatory agent.

As used herein, a “patient” means an animal, e.g., mammal, including ahuman. The patient may be diagnosed with an immune disease. “Treatment”or “treat” or “treating” refers to the process involving alleviating theprogression or severity of a symptom, disorder, condition, or disease.An “immune disease” refers to any disease associated with thedevelopment of an immune reaction in an individual, including a cellularand/or a humoral immune reaction. Examples of immune diseases include,but are not limited to, inflammation, allergy, autoimmune disease, orgraft-related disease. Thus, the patient may be diagnosed with anautoimmune disease or inflammatory disease. An “autoimmune disease”refers to any disease associated with the development of an autoimmunereaction in an individual, including a cellular and/or a humoral immunereaction. An example of an autoimmune disease is inflammatory boweldisease (IBD), including, but not limited to ulcerative colitis andCrohn's disease. Other autoimmune diseases include systemic lupuserythematosus, multiple sclerosis, rheumatoid arthritis, diabetes,psoriasis, scleroderma, and atherosclerosis. Graft-related diseasesinclude graft versus host disease (GVHD), acute transplantationrejection, and chronic transplantation rejection.

Diseases that can be treated by administering the antibody of thedisclosure may be selected from the group consisting of Addison'sdisease, allergies, anaphylaxis, ankylosing spondylitis, asthma,atherosclerosis, atopic allergy, autoimmune diseases of the ear,autoimmune diseases of the eye, autoimmune hepatitis, autoimmuneparotitis, bronchial asthma, coronary heart disease, Crohn's disease,diabetes, epididymitis, glomerulonephritis, Graves' disease,Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia,idiopathic thrombocytopenic purpura, inflammatory bowel disease, immuneresponse to recombinant drug products (e.g., Factor VII inhemophiliacs), lupus nephritis, systemic lupus erythematosus, multiplesclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever,rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, transplant rejection, vasculitis,and ulcerative colitis.

The pharmaceutical composition may be administered alone or as acombination therapy, (i.e., simultaneously or sequentially) with animmunosuppressive/immunomodulatory and/or anti-inflammatory agent.Different immune diseases can require use of specific auxiliarycompounds useful for treating immune diseases, which can be determinedon a patient-to-patient basis. For example, the pharmaceuticalcomposition may be administered in combination with one or more suitableadjuvants, e.g., cytokines (IL-10 and IL-13, for example) or otherimmune stimulators, e.g., chemokines, tumor-associated antigens, andpeptides. Suitable adjuvants are known in the art.

Any suitable method or route can be used to administer the antibody, orantigen binding portion thereof, or the pharmaceutical composition.Routes of administration include, for example, intravenous,intraperitoneal, subcutaneous, or intramuscular administration. Atherapeutically effective dose of administered antibody depends onnumerous factors, including, for example, the type and severity of theimmune disease being treated, the use of combination therapy, the routeof administration of the antibody, or antigen binding portion thereof,or pharmaceutical composition, and the weight of the patient. Anon-limiting range for a therapeutically effective amount of a domainantibody is 0.1-20 milligram/kilogram (mg/kg), and in an aspect, 1-10mg/kg, relative to the body weight of the patient.

Kits

A kit useful for treating an immune disease in a human patient isprovided. A kit useful for treating or preventing an autoimmune diseaseor inflammatory disease in a human patient is also provided. The kit cancomprise (a) a dose of an antibody, or antigen binding portion thereof,of the present disclosure and (b) instructional material for using theantibody, or antigen binding portion thereof, in the method of treatingan immune disease, or for using the antibody, or antigen binding portionthereof, in the method of treating or preventing an autoimmune orinflammatory disease, in a patient.

“Instructional material,” as that term is used herein, includes apublication, a recording, a diagram, or any other medium of expression,which can be used to communicate the usefulness of the compositionand/or compound of the invention in a kit. The instructional material ofthe kit may, for example, be affixed to a container that contains thecompound and/or composition of the invention or be shipped together witha container, which contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

EXAMPLES Example 1: Binding of Mouse Anti-Human CD40 Antibodies to HumanCD40

Mouse anti-human-CD40 antibodies were generated and were tested forbinding to human CD40 by surface plasmon resonance (SPR). The Vh and Vksequences for each antibody are shown in Table 8.

TABLE 8 Mouse anti-human-CD40 variable heavy and light sequences IDVH Sequence VL Sequence ADX_Y1060.ZZ0-1-Vh ADX_Y1060.ZZ0-1-VkADX_Y1060.ZZ0-1 QVQLVQSGAEVKKPGASVKVSCKASG DIQMTQSPSSVSASVGDRVTITCRASQYTFTGYYMHWVRQAPGQGLEWMGWIN GIYSWLAWYQQKPGKAPNLLIYTASTLPDSGGTNYAQKFQGRVTMTRDTSIST QSGVPSRFSGSGSGTDFTLTISSLQPEAYMELNRLRSDDTAVYYCARDQPLGY DFATYYCQQANIFPLTFGGGTKVEIKCTNGVCSYFDYWGQGTLVTVSS (SEQ ID NO: 76) (SEQ ID NO: 53)ADX_Y1072.ZZ0-1-Vh ADX_Y1072.ZZ0-1-Vk ADX_Y1072.ZZ0-1QVQFQQSGAELARPGASVKLSCKASG DVVMTQTPLSLPVSLGDQASISCRSSQYTFTSYWMQWVKQRPGQGLEWIGTIY SLVHRNGNTYLHWYLQKPGQSPKLLIYPGDGDSRYNQKFKGKALLTADKSSSI RVSNRFSGVPDRFSGSGSGTDFTLKISAYMQLNSLASEDSAVYFCARFSLYDG RVEAEDLGIYFCSQSTHFPYTFGGGTKYPYYFDYWGQGTTLTVSS LEIK (SEQ ID NO: 54) (SEQ ID NO: 77)ADX_Y1234.ZZ0-1-Vh ADX_Y1234.ZZ0-1-Vk ADX_Y1234.ZZ0-1EVQLVESGGGLVKPGGSLKLSCAASG DILLTQSPAILSVSPGERVSFSCRASQFAFSSYDMSWVRQTPEKRLEWVAYIN SIGTSIHWYQQRTIGSPRLLIKYASESSGVGNTYYPDTVKGRFTISRDNAKNT ISGIPSRFSGSGSGTDFTLSINSVESELYLQMSSLKSEDTAMYYCARHGNYAW DIADYYCQQINSWPLTFGAGTKLELK FAYWGQGTLVTVSA(SEQ ID NO: 78) (SEQ ID NO: 55) ADX_Y1236.ZZ0-1-Vh ADX_Y1236.ZZ0-1-VkADX_Y1236.ZZ0-1 DVQLVESGGGLVQPGGSRKLSCAASG DIVMTQSQKFMSTSVGDRISITCKASQFTFSSFGMHWVRQAPEKGLEWVAYIS NVRTAVAWYQQKPGQSPKALIYLASNRSGSSTIYYADTVKGRFTISRDNPKNT HTGVPARFSGSGSGTSYSLTISRMEAELFLQMTSLRSEDTAMYYCARYGNYAM DAATYYCQQRSSYPLTFGAGTKLELK DYWGQGTSVTVSS(SEQ ID NO: 79) (SEQ ID NO: 56) ADX_Y1238.ZZ0-1-Vh ADX_Y1238.ZZ0-1-VkADX_Y1238.ZZ0-1 QVQLQQSGAELVRPGTSVKVSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYAFTNYLIEWVKQRPGQGLEWIGVIN DVRTGVAWYQQKPGQSPKLLIYSASYRPGSGGTNYNEKFKGKATLTADKSSST NTGVPDRFTGSRSGTDFTFTISSVQAEAYMQLSSLTSDDSAVYFCARSQLGRR DLAVYYCQQHYSPPYTFGGGTKLEIK FDYWGQGTTLTVSS(SEQ ID NO: 80) SEQ ID NO: 57) ADX_Y1241.ZZ0-1-Vh ADX_Y1241.ZZ0-1-VkADX_Y1241.ZZ0-1 EFQLQQSGPELVKPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYTFTNYIIQWVKKQPGQGLEWIGYIN DVGTAVAWYQQKPGQSPKLLIYWASTRPYSSETNYNEKFKGKATLTSDKSSST HTGVPDRFTGSGSGTDFTLTISNVQSEAYMELSSLTSEDSAIYFCARDLIGNY DLADYFCQQYSSYPLTFGAGTKLELK WGQGTTLTVSS(SEQ ID NO: 81) (SEQ ID NO: 58) ADX_Y1242.ZZ0-1-Vh ADX_Y1242.ZZ0-1-VkADX_Y1242.ZZ0-1 EFQLQQSGPELVKPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYVMHWVKQKPGQALEWIGYIN DVSTAVAWYQQKPGQSPKLLIYSASYRPSNDGSEYNERFKGKATLTSDKSSTT YTGVPDRFTGSGSGTDFTFTISSVQAEAYMELSSLTSEDSAVYYCARWAPYPF DLAVYYCQQHYSTPYTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 82) (SEQ ID NO: 59) ADX_Y1249.ZZ0-1-Vh ADX_Y1249.ZZ0-1-VkADX_Y1249.ZZ0-1 QVQLQQSGAELARPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYTFTSYTMHWVKQRPGQGLEWIGYID DVSTAVAWYQQKPGQSPKLLIYSASYRPSSHYTNYNQKFKGTATLTADKSSNT YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLSSLTSEDSAVYYCARDYRYAY DLAVYYCQQHYSTPWTFGGGTKLEIK WYFDVWGAGTTLTVSS(SEQ ID NO: 83) (SEQ ID NO: 60) ADX_Y1256.ZZ0-1-Vh ADX_Y1256.ZZ0-1-VkADX_Y1256.ZZ0-1 QVQLQQSGAELAKPGSSVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYAFTSYWMHWVKQRPGQGLEWIGYIN DVSTAVAWYQQKPGQSPKLLIYSASYRPTTGYSAYNQKFKDKATLTADKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYLQLTSLTSEDSAVYFCSRWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 84) (SEQ ID NO: 61) ADX_Y1257.ZZ0-1-Vh ADX_Y1257.ZZ0-1-VkADX_Y1257.ZZ0-1 QVQLQQSGAELAKPGSSVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYAFTSYWMHWVKQRPGQGLEWIGYIN DVSTAVAWYQQKPGQSPKLLIYSASYRPTTGYSAYNQKFKAKTTLTADKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLTSLTFEDSAVYFCSRWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 85) (SEQ ID NO: 62) ADX_Y1258.ZZ0-1-Vh ADX_Y1258.ZZ0-1-VkADX_Y1258.ZZ0-1 QVQLQQSGAELAKPGSSVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYAFTSYWMHWIKQRPGQGLEWIGFIN DVSTAVAWYQQKPGQSPKLLIYSASYRPTTGYSEYNQKFKDKATLTADKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLNSLTSEDSAVYFCARWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 86) (SEQ ID NO: 63) ADX_Y1259.ZZ0-1-Vh ADX_Y1259.ZZ0-1-VkADX_Y1259.ZZ0-1 QVQLQQSGAELAKPGASVKMSCKTSG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWMHWIKQRPGQGLEWIGFIN DVSTAVAWYQQKPGQSPKLLIYSASYRPTTGYTEYNQKFKDKATLTADKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLSSLSSEDSAVYYCSRWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 87) (SEQ ID NO: 64) ADX_Y1260.ZZ0-1-Vh ADX_Y1260.ZZ0-1-VkADX_Y1260.ZZ0-1 QVQLQQSGAELTKPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWMHWVKQRPGQGLEWIGSIN DVSTAVAWYQQKPGQSPKLLIYSASYRPSTGYTEDNQKFKDKATLTADKSSTT YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLSSLTSEDSAVYYCARWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 88) (SEQ ID NO: 65) ADX_Y1261.ZZ0-1-Vh ADX_Y1261.ZZ0-1-VkADX_Y1261.ZZ0-1 QVQLQQSGAERAKPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWMHWIKQRPGQGLEWIGFIN DVSTAVAWYQQKPGQSPKLLIYSASYRPNTGHTDYNQKFKDKATLTADKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLSSLTSEDSAVYFCSRWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 89) (SEQ ID NO: 66) ADX_Y1262.ZZ0-1-Vh ADX_Y1262.ZZ0-1-VkADX_Y1262.ZZ0-1 QVQLQQSGAELAKPGSSVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYAFTSYWMHWVKQRPGQGLEWIGYIN DVSTAVAWYQQKPGQSPKLLIYSASYRPTTGYSAYNQKFKDKATLTADKSSST YTGVPDRFTGSGYGTDFTFTISSVQAEAYMQLNSLTSEDSAVYYCARWDPRPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 90) (SEQ ID NO: 67) ADX_Y1263.ZZ0-1-Vh ADX_Y1263.ZZ0-1-VkADX_Y1263.ZZ0-1 QVQLQQSGAELAKPGTSVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWVHWVKERPGQGLEWIGHTN DVSTAVAWYQQKPGQSPKLLIYSASYRPNTGYTEYNQKFKDKATLTVDRSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMQLNSLTSEDSAVYYCARWDPRPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 91) (SEQ ID NO: 68) ADX_Y1264.ZZ0-1-Vh ADX_Y1264.ZZ0-1-VkADX_Y1264.ZZ0-1 EVQLQQSGTVLARPGASVKMSCRASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFSSYWMHWVKQRPGQGLEWIGSIN DVSTAVAWYQQKPGQSPKLLIYSASYRPGNSDAFYNQQFKGKAKLTAVTSAST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMELSSLTNEDSAVYYCTRWGLPPF DLAVYYCHQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 92) (SEQ ID NO: 69) ADX_Y1265.ZZ0-1-Vh ADX_Y1265.ZZ0-1-VkADX_Y1265.ZZ0-1 EVQLQQSGTVLAGPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWMHWVKQRPGQDLEWIGTIN DVSTAVAWYQQKPGQSPKLLIYSASYRPGKGDSNYNQKFKGKAKLTAVTSAST YTGVPDRFTGSGSGTDFTFTISSVQAEAYMELSSLTNEDSAVYYCTRWGLPPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 93) (SEQ ID NO: 70) ADX_Y1266.ZZ0-1-Vh ADX_Y1266.ZZ0-1-VkADX_Y1266.ZZ0-1 QVQLQQPGAELVKPGASVRLSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWMHWVKQRPGQGLEWIGQIN DVSTAVAWYQQKPGQSPKLLIYSASYRPSNGRTQYNEKFKSMATLTVDKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYIQLSSLTSEDSAVYYCARWGLQPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 94) (SEQ ID NO: 71) ADX_Y1267.ZZ0-1-Vh ADX_Y1267.ZZ0-1-VkADX_Y1267.ZZ0-1 QVQLQQPGAELVKPGASVRLSCEASG DIVMTQSHKFMSTSVGDRVSITCKASQYSFTSYWMHWVKQRPGQGLEWIGQIN DVSTAVAWYQQKPGQSPKLLIYSASYRPSNGRTQYNEKFKSMATLTVDKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYIQLNSLTSEDSAVYYCARWGLQPF DLAVYYCLQHYTTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 95) (SEQ ID NO: 72) ADX_Y1268.ZZ0-1-Vh ADX_Y1268.ZZ0-1-VkADX_Y1268.ZZ0-1 QVQLQQPGAELVKPGASVRLSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYAFTSYWMHWVKQRPGQGLEWIGQIN DVSTAVAWYQQKPGQSPKLLIYSASYRPSNGRSQYNEKFKTMATLTVDKSSST YTGVPDRFTGSGSGTDFTFTISSVQAEAYIQLSSLTSEDSAVYYCARWGLQPF DLAVYYCQQHYSTPWTFGGGTKLEIK AYWGQGTLVTVSA(SEQ ID NO: 96) (SEQ ID NO: 73) ADX_Y1269.ZZ0-1-Vh ADX_Y1269.ZZ0-1-VkADX_Y1269.ZZ0-1 QVQLQQSGAELPRPGASVKMSCKASG DIVMTQSHKFMSTSVGDRVSITCKASQYTFTDYTVHWVKQRPGQGLEWIGYIN DVSPNVAWYQQKPGQSPKLLIYSTSYRPSSSYTSYDQKFKDKATVTADKSSST YTGVPDRFTGSRSGTDFTFTISSVQAEAYMQLSSLTSEDSAVYYCARRTMYWY DLAIYYCQQHYSTPLTFGAGTKLELK FDIWGAGTTVTVSS(SEQ ID NO: 97) (SEQ ID NO: 74) ADX_Y1297.ZZ0-1-Vh ADX_Y1297.ZZ0-1-VkADX_Y1297.ZZ0-1 QVQLQQSGAELVKPGASVKLSCKASG DIVMTQSHKFMSTSVGDRVSVTCKASQYTFTSYWMHWVKQRPGQGLEWIGEID NVRINVAWYQQKPGQSPKALIYSASYRPSDSYTNYNQNFKGKATLTVDKSSST YSGVPDRFTGSGSGTDFTLTITNVQSEAYMQLSSLTSEDSAVYYCARETYYYG DLAEYFCQQYNTYPLTFGAGTKLELK SRFPYWGQGTLVTVSA(SEQ ID NO: 98) (SEQ ID NO: 75)

The CD40 kinetic and affinity data for human-CD40 monomer binding tomouse anti-human CD40 antibodies captured on a protein A sensor chipsurface were assessed by SPR. The data are shown in Table 9. The datashown are for a single concentration of CD40 analyte (1 μM) and aretherefore reported as apparent (app) values.

TABLE 9 SPR kinetic/affinity data Antibody ka,_(app) (1/Ms) kd,_(app)(1/s) KD_(app) (M) ADX_Y1072.ZZ0-1 7.7E+04 9.2E−03 1.2E−07ADX_Y1238.ZZ0-1 5.5E+04 1.2E−04 2.2E−09 ADX_Y1258.ZZ0-1 1.7E+04 1.3E−047.9E−09 ADX_Y1260.ZZ0-1 5.2E+04 2.1E−04 4.0E−09 ADX_Y1262.ZZ0-1 3.7E+052.5E−03 6.6E−09 ADX_Y1264.ZZ0-1 1.4E+04 2.3E−04 1.7E−08 ADX_Y1267.ZZ0-13.7E+05 4.1E−04 1.1E−09 ADX_Y1268.ZZ0-1 3.2E+05 4.6E−04 1.4E−09

Based on the SPR data and sequence data, three antibodies,ADX_Y1258.ZZ0-1, ADX_Y1262.ZZ0-1, and ADX_Y1268.ZZ0-1, were selected forhumanization.

Example 2: Humanization and Selection of Humanized Variants of Y12XX

Humanization background/procedure is as discussed in section “II.Engineered and Modified Antibodies” in WO2017004006, which isincorporated herein by reference in its entirety. Based on thisanalysis, nine (9) humanized Vh sequences (Vh-hz1, Vh-hz2, Vh-hz3,Vh-hz4, Vh-hz5, Vh-hz6, Vh-hz9, Vh-hz10, and Vh-hz11) and three (3)humanized Vκ sequences (Vk-hz1, Vk-hz2, and Vk-hz3), were selected fortesting. In addition, five (5) humanized Vh sequences (Vh-hz7, Vh-hz8,Vh-hz12, Vh-hz13, and Vh-hz14) were designed to contain mutationsintended to reduce chemical liability risk designed. The mutationsinclude D100Q (Y1262_IGHV1.6908-D100Q) and P101A(Y1262_IGHV1.6908-P101A) mutations to mitigate potential hydrolysis riskin Y1262_IGHV1.6908. The mutations also include N55Q(Y1268_IGHV1.6908-N55Q), G56A (Y1268_IGHV1.6908-G56A), and the S54T-N55Tdouble mutation (Y1268_IGHV1.6908-S54T-N55T) to mitigate potentialdeamidation risk in Y1268_IGHV1.6908. The S54T-N55T double mutation wasdesigned based on the corresponding amino acid residues found at thesepositions in ADX_Y1262.ZZ0-1-Vh. See Table 10.

The sequences for these variants are shown in Table 10.

TABLE 10 Variable SEQ ID Hz # ID NO:  Sequence Y1258-Vh Vh-C1  99QVQLQQSGAELAKPGSSVKMSCKASGYAFTSYWMHWIKQRPGQGLEWIGFINPTTGYSEYNQKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYFCARWGLPPFAYWGQGT LVTVSA Y1258_IGHV1.6908-Vh Vh-hz1100 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV RQAPGQGLEWMGFINPTTGYSEYNQKFKDRVTITADK STSTAYMELSSLRSEDTAVYYCARWGLPPFAYWGQGT LVTVSS Y1258_IGHV1.6908_Vh-hz2 101 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMH WV A40R-VhRQRPGQGLEWMGFINPTTGYSEYNQKFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLPPFAYWGQGT LVTVSS Y1258_IGHV1.6908_ Vh-hz3102 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV A40R-M48I-S84N-VhRQRPGQGLEWIGFINPTTGYSEYNQKFKDRVTITADKSTSTAYMELNSLRSEDTAVYYCARWGLPPFAYWGQGT LVTVSS Y1262-Vh Vh-C2 103QVQLQQSGAELAKPGSSVKMSCKASGYAFTSYWMHWVKQRPGQGLEWIGYINPTTGYSAYNQKFKDKATLTADKSSSTAYMQLNSLTSEDSAVYYCARWDPRPFAYWGQGT LVTVSA Y1262-IGHV1.6908_Vh Vh-hz4104 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWVRQAPGQGLEWMGYINPTTGYSAYNQKFKDKATLTADKSTSTAYMELSSLRSEDTAVYYCARWDPRPFAYWGQGT LVTVSS Y1262-IGHV1.6908_ Vh-hz5105 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV A40R-VhRQRPGQGLEWMGYINPTTGYSAYNQKFKDKATLTADKSTSTAYMELSSLRSEDTAVYYCARWDPRPFAYWGQGT LVTVSS Y1262_IGHV1.6908_ Vh-hz6106 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV A40R-M48I-S84N-VhRQRPGQGLEWIGYINPTTGYSAYNQKFKDKATLTADKSTSTAYMELNSLRSEDTAVYYCARWDPRPFAYWGQGT LVTVSS Y1262_IGHV1.6908- Vh-hz7107 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV D100Q-VhRQAPGQGLEWMGYINPTTGYSAYNQKFKDKATLTADKSTSTAYMELSSLRSEDTAVYYCARWQPRPFAYWGQGT LVTVSS Y1262_IGHV1.6908- Vh-hz8108 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV P101A-VhRQAPGQGLEWMGYINPTTGYSAYNQKFKDKATLTADKSTSTAYMELSSLRSEDTAVYYCARWDARPFAYWGQGT LVTVSS Y1268-Vh Vh-C3 109QVQLQQPGAELVKPGASVRLSCKASGYAFTSYWMHWVKQRPGQGLEWIGQINPSNGRSQYNEKFKTMATLTVDKSSSTAYIQLSSLTSEDSAVYYCARWGLQPFAYWGQGT LVTVSA Y1268_IGHV1.6908-Vh Vh-hz9110 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWVRQAPGQGLEWMGQINPSNGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLQPFAYWGQGT LVTVSS Y1268_IGHV1.6908_ Vh-hz10111 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV A40R-VhRQRPGQGLEWMGQINPSNGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLQPFAYWGQGT LVTVSS Y1268_IGHV1.6908_ Vh-hz11112 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV A40R-M48I-VhRQRPGQGLEWIGQINPSNGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLQPFAYWGQGT LVTVSS Y1268_IGHV1.6908- Vh-hz12113 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV N55Q-VhRQAPGQGLEWMGQINPSQGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLQPFAYWGQGT LVTVSS Y1268_IGHV1.6908- Vh-hz14  4 QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWV S54T-N55T-VhRQAPGQGLEWMGQINPSNARSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLQPFAYWGQGT LVTVSS Y1268-Vk Vk-C1 114QVQLVQSGAEVKKPGSSVKVSCKASGYAFTSYWMHWVRQAPGQGLEWMGQINPTTGRSQYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGLQPFAYWGQGT LVTVSS Y1258-Vk Vk-C1 114DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFT ISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKY1262-Vk Vk-C2 115 DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGYGTDFTFT ISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIKY1258_IGKV1.3301-Vk Vk-hz1 116 DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFT ISSLQPEDIATYYCQQHYSTPWTFGGGTKVEIKY1258_IGKV1.3902_Vk Vk-hz2  10 DIQMTQSPSFLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLT ISSLQPEDFATYYCQQHYSTPWTFGGGTKVEIKY1258_IGKV3.1501-Vk Vk-hz3  16 EIVMTQSPATLSVSPGERATLSCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGIPARFSGSGSGTEFTLT ISSLQSEDFAVYYCQQHYSTPWTFGGGTKVEIK

Table 10 provides the heavy and variable light domain sequences used forconstruction of humanized antibodies, as well as chimeric antibodycontrols, for CD40 binding analyses using BIAcore™ surface plasmonresonance (SPR), as well as Octet BLI titer analyses (discussed below).

Vh sequences were formatted with IgG1-P238K isotype (CH1-IgG1-P238K; SEQID NO:25). Vκ sequences were formatted as a full light chain with acommon CL sequence (amino acids 108-214 of SEQ ID No: 11). In Table 11,“Y1258” and “Y1262” refer to chimeric molecules containing mousevariable regions and human constant regions. The various differentcombinations of the humanized HC constructs and LC constructs, as wellas the chimeric Y1258 and Y1262 molecules were expressed as 3 milliliter(ml) supernatants for titer analysis and CD40 binding analysis. Thefamily of molecules was collectively identified with an “Y12XX” prefix,followed by a “hz #” suffix to uniquely identify different heavychain/light chain pairs.

Titer analysis was performed using Biolayer Interferometry (BLI) on anOctet RED instrument (Fortebio) by capturing antibodies from supernatantusing protein A sensor tips and measuring capture response with respectto a standard curve obtained using a control antibody sample. SPR datawere obtained by capturing antibodies on a protein A surface and testingthe binding of 500 nM and 50 nM injections of human-CD40 analyte, usinga BIAcore™ T200 instrument (GE Healthcare). The kinetic data for the twoconcentrations of hCD40-monomer were fit to a 1:1 Langmuir model, toyield estimates of the kinetic and affinity values for theseinteractions, and for comparison of the different molecules.

The Octet titer and BIAcore™ SPR CD40 binding data are provided in Table11. In addition to testing supernatant (“sup”) samples, purifiedchimeric Y1258, Y1262 and Y1268 antibodies containing human wild-typeIgG1f isotype

(ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; SEQ ID NO: 117)were tested by SPR as controls; these are named “Y1258-hIgG1f”,“Y1632-hIgG1f” and “Y1268-hIgG1f” in Table 11, and the Vh and Vk chainsare denoted as “Chim-P.”

TABLE 11 Octet titer and BIAcore ™ SPR CD40 binding data Titer ka kd KDAntibody ID Vh Vk Sample (μg/ml) (1/Ms) (1/s) (M) Y1258-hIgG1f Chim-PChim-P purified n/a 5.8E+04 5.9E−07 1.0E−11 Y1258 Vh-C1 Vk-C1 sup 54.25.6E+04 3.8E−06 6.7E−11 Y12XX-hz1 Vh-hz1 Vk-hz1 sup 3.8 1.5E+04 1.6E−051.1E−09 Y12XX-hz15 Vh-hz1 Vk-hz2 sup 34.6 1.6E+04 1.3E−04 8.0E−09Y12XX-hz29 Vh-hz1 Vk-hz3 sup 56.7 3.7E+04 2.6E−06 6.9E−11 Y12XX-hz2Vh-hz2 Vk-hz1 sup 4.5 1.7E+04 7.1E−05 4.1E−09 Y12XX-hz16 Vh-hz2 Vk-hz2sup 58.6 2.0E+05 6.4E−04 3.2E−09 Y12XX-hz30 Vh-hz2 Vk-hz3 sup 82.64.1E+04 7.3E−07 1.8E−11 Y12XX-hz3 Vh-hz3 Vk-hz1 sup 6.4 1.5E+04 6.7E−054.5E−09 Y12XX-hz17 Vh-hz3 Vk-hz2 sup 50.7 3.7E+05 7.7E−02 2.1E−07Y12XX-hz31 Vh-hz3 Vk-hz3 sup 93.9 4.1E+04 2.9E−07 7.0E−12 Y1262-hIgG1fChim-P Chim-P purified n/a 4.8E+05 5.5E−03 1.2E−08 Y1262 Chim Chim sup92.2 3.5E+05 2.8E−03 8.0E−09 Y12XX-hz4 Vh-hz4 Vk-hz1 sup 4.7 4.6E+052.2E−03 4.7E−09 Y12XX-hz18 Vh-hz4 Vk-hz2 sup 73.6 3.5E+05 2.4E−037.1E−09 Y12XX-hz32 Vh-hz4 Vk-hz3 sup 104.3 2.9E+05 3.0E−03 1.0E−08Y12XX-hz5 Vh-hz5 Vk-hz1 sup 4.5 3.5E+05 2.5E−03 7.2E−09 Y12XX-hz19Vh-hz5 Vk-hz2 sup 56.7 3.8E+05 2.3E−03 6.2E−09 Y12XX-hz33 Vh-hz5 Vk-hz3sup 85.5 2.9E+05 3.3E−03 1.1E−08 Y12XX-hz6 Vh-hz6 Vk-hz1 sup 6.7 3.8E+052.4E−03 6.4E−09 Y12XX-hz20 Vh-hz6 Vk-hz2 sup 50.3 3.1E+05 2.5E−038.2E−09 Y12XX-hz34 Vh-hz6 Vk-hz3 sup 93.7 3.7E+05 2.8E−03 7.6E−09Y12XX-hz8 Vh-hz8 Vk-hz1 sup 11.2 7.2E+05 1.5E−01 2.1E−07 Y12XX-hz22Vh-hz8 Vk-hz2 sup 49.1 3.7E+05 7.9E−02 2.1E−07 Y12XX-hz36 Vh-hz8 Vk-hz3sup 136.7 3.9E+05 1.0E−01 2.5E−07 Y1268-hIgG1f Chim-P Chim-P purifiedn/a 4.0E+05 1.3E−03 3.2E−09 Y12XX-hz9 Vh-hz9 Vk-hz1 sup 5.1 2.0E+058.9E−04 4.6E−09 Y12XX-hz23 Vh-hz9 Vk-hz2 sup 59.4 2.0E+05 6.4E−043.2E−09 Y12XX-hz37 Vh-hz9 Vk-hz3 sup 138.4 2.6E+05 8.4E−04 3.3E−09Y12XX-hz10 Vh-hz10 Vk-hz1 sup 8.6 2.0E+05 7.3E−04 3.6E−09 Y12XX-hz24Vh-hz10 Vk-hz2 sup 48.1 1.9E+05 8.2E−04 4.4E−09 Y12XX-hz38 Vh-hz10Vk-hz3 sup 185.5 2.5E+05 8.8E−04 3.6E−09 Y12XX-hz11 Vh-hz11 Vk-hz1 sup7.5 1.8E+05 8.8E−04 5.0E−09 Y12XX-hz25 Vh-hz11 Vk-hz2 sup 55.4 1.9E+056.4E−04 3.4E−09 Y12XX-hz39 Vh-hz11 Vk-hz3 sup 134.2 2.4E+05 8.4E−043.5E−09 Y12XX-hz12 Vh-hz12 Vk-hz1 sup 2.7 1.7E+05 1.4E−03 8.3E−09Y12XX-hz26 Vh-hz12 Vk-hz2 sup 36.8 1.6E+05 1.2E−03 7.5E−09 Y12XX-hz40Vh-hz12 Vk-hz3 sup 99.8 2.4E+05 1.1E−03 4.7E−09 Y12XX-hz13 Vh-hz13Vk-hz1 sup 3.0 2.1E+05 8.3E−04 3.9E−09 Y12XX-hz27 Vh-hz13 Vk-hz2 sup49.5 1.9E+05 8.8E−04 4.7E−09 Y12XX-hz41 Vh-hz13 Vk-hz3 sup 52.7 2.5E+059.4E−04 3.8E−09 Y12XX-hz14 Vh-hz14 Vk-hz1 sup 5.0 1.7E+05 8.3E−045.0E−09 Y12XX-hz28 Vh-hz14 Vk-hz2 sup 70.1 1.8E+05 6.2E−04 3.5E−09Y12XX-hz42 Vh-hz14 Vk-hz3 sup 100.0 2.4E+05 8.3E−04 3.5E−09

For a given heavy chain construct, the titer is generally highest whenpaired with light chains containing Vk-hz3 (SEQ ID NO:18), lower forheavy chains paired with Vk-hz2 (SEQ ID NO:10), and the lowest for heavychains paired with Vk-hz1 (SEQ ID NO:116) containing light chains.

The SPR analysis data show that the antibodies bound with variableaffinity to CD40, with KD values ranging from greater than 1 E-07 toless than 1 E-09. For some antibodies, the affinity was too strong toaccurately determine with confidence in this assay, because thedissociation rate was too slow to measure. These values, which areitalicized in the table, are beyond the limit of accurate quantitationin this assay.

Based on the sequences, titer, and SPR binding data, antibodies wereselected for larger scale expression, purification, and furthercharacterization. SPR analysis using purified antibodies was performedby capturing antibodies on a protein A surface, with binding of a500-3.9 nM (2:1) dilution series of human-CD40 monomer, at either 25° C.or 37° C. in PBS-T pH 7.1 buffer; the titration data was fit to a 1:1Langmuir model. The data is provided in Table 12.

TABLE 12 SPR kinetic/affinity data 25° C. 37° C. ka kd KD ka kd KDLigand Sample (1/Ms) (1/s) (nM) (1/Ms) (1/s) (nM) Y12XX-hz28 hCD402.2E+05 6.9E−04 3.1 4.4E+05 3.7E−03 8.5 Y12XX-hz40 hCD40 2.9E+05 1.3E−034.4 5.2E+05 5.7E−03 10.9 Y12XX-hz42 hCD40 3.1E+05 7.3E−04 2.3 6.3E+053.4E−03 5.5 Antibody B 6.1E+04 2.3E−03 37

These data show that the selected Y12XX antibodies bind with highaffinity and KD values in the range of KD=1 E-9 M at 25° C. The bindingis compared to that of another anti-CD40 antibody, antibody BI-mAb-B(U.S. Pat. No. 9,090,696, heavy chain sequence SEQ ID NO: 32 and lightchain sequence SEQ ID NO: 31; referred to herein as “Antibody B” and“BI-LALA”). As shown by the data in Table 12, Antibody B binds to CD40with much lower affinity than the humanized Y12XX molecules.

All three humanized versions of the Y12XX antibody were potentantagonists of B cell proliferation stimulated with CD40L-IZ trimericagonist. See Table 13.

TABLE 13 Inhibition of B cell proliferation induced by soluble CD40Ltrimer Average (IC50 Standard Deviation ng/ml) (STDEV) n donors AntibodyB 9.4 3.9 6 Y12XX-hz28-P238K 6.7 3.6 8 Y12XX-hz40-P238K 6.0 4.7 2Y12XX-hz42-P238K 12.1 2.3 2

Y12XX-hz28-P238K was also a potent antagonist of B cell proliferationstimulated with cellular CD40L from CD40L-expressing CHO cells. SeeTable 14.

TABLE 14 Potency for inhibition of CD40L expressing CHO cellsstimulation of B cell proliferation Potency (IC50 ng/ml Standard n of %inhibition) deviation donors Antibody B 62% * 25% 6 Y12XX-hz28-P238K38.1 9.8 8 * % inhibition at highest dose tested (1-3 μg/ml)

The data for the humanized Y12XX antibodies is compared to that ofAntibody B, which showed potent inhibition of B cell proliferationdriven by soluble CD40L signals, but was much less effective atinhibition of B cell proliferation driven by cellular CD40L (CHO cellsoverexpressing CD40L). In contrast, humanized Y12XX antibodies exhibitedonly a <10 fold shift in the potency for inhibition of cell surfaceCD40L stimulation, providing more robust blockade of B cell responses toCD40L.

Humanized Y12XX antibodies were formatted with IgG1-P238K isotype(CH1-IgG1-P238K; SEQ ID NO: 25) to reduce the binding affinity for FcγRsand reduce FcγR-mediated signaling. FcγR binding for a representativehumanized Y12XX antibody with this IgG1-P238K isotype(Y12XX-hz28-IgG1-P238K) was compared to the binding of a controlantibody formatted with a wild type IgG1 isotype (control-IgG1) as wellas Antibody B which has an IgG1 isotype containing the mutationsL234A-L235A. These L234A-L235A mutations are also introduced to reduceFcγR binding.

FcγR binding SPR studies were performed by capturing antibodies on aprotein A sensor chip surface and binding purified His-tagged humanFcγRs as analyte. hCD64 binding consisted of a titration of 10 μM-1.5 nMhCD64 (2:1 dilution series), while data for the low affinity FcγRshCD32a-H131, hCD32a-R131, hCD32b, hCD16a-V158, and hCD16a-F158 consistedof a titration of 10 uM-13.7 nM FcγR protein.

The control-IgG1 antibody demonstrated binding to all of the FcγRstested. See FIG. 1A. Compared to wild type, the Y12XX-hz28-IgG1-P238Kantibody demonstrated 125-fold weaker binding to hCD64, and demonstratedno detectable binding to any of the low affinity FcγRs hCD32a-H131,hCD32a-R131, hCD32b, hCD16a-V158 and hCD16a-F158 tested. See FIG. 1B.Antibody B also demonstrated weaker hCD64 binding than wild type IgG1,but also demonstrated appreciable binding to hCD16a-V158 (KD=7 μM) andsome weak binding to hCD32a-H131 and hCD32a-R131. See FIG. 1C. The KDvalues are provided in FIG. 1D.

Humanized versions of the antibody Y12XX with P238K mutation in the Fcregion were further tested for any agonist activity. Monocyte derivedimmature dendritic cells (iDC) are very sensitive to CD40 activation,increasing cytokine production (IL-6) and upregulating surface markersof activation (CD86 and CD54) upon CD40 stimulation. Therefore, the mostpromising humanized Y12XX antibodies were tested to assess their abilityto stimulate iDC. The ability of CD40 antibodies to agonize CD40 can beenhanced by clustering or cross-linking binding of the Fc portions ofthe molecule to cell surface FcγR. Addition of CHO cells highlyover-expressing CD32a, the low affinity FcγR, were used to evaluate thepotential for FcγR mediated clustering/cross-link. The ratio of CHOcells to iDCs was 1:6 in these experiments, representing a potentiallyexaggerated level of clustering/cross-linking. BMS-986090 and 2141 wereused as positive controls. BMS-986090 is an anti-CD40 antagonist domainantibody fused to IgG4 Fc (see SEQ ID NO: 1287 in WO 2012/145673). 2141(mAb 134-2141) is a partial CD40 agonist (see Robert Vonderheide et al.,2007, J. Clin. Oncol. 25(7): 876-883). L6-IgG4 is a fusion protein withno CD40 binding capability, and served as a negative control.

As illustrated by the data in FIGS. 2A-2C, the addition of either thepartial agonist 2141 or BMS-986090 led to only weak activation of iDC ina subset of donors. However, addition of CD32a-expressing CHO cells toeither 2141 or BMS-986090 led to robust increases in IL-6 production(FIG. 2B) and CD86 and CD54 upregulation (FIG. 2B and FIG. 2C,respectively) in nearly every donor tested, consistent with FcγRmediated clustering of these molecules through their Fc portions leadingto CD40 activation. In contrast, Y12XX-hz28-P238K and Y12XX-hz40-P238Keither alone or with CD32 dependent clustering did not show any signs ofiDC activation above that observed with the negative control using iDCcells from 6-10 donors. Y12XX-hz42-P238K was tested in cells from 4donors and exhibited signs of weak activation including IL-6 productionand CD86 and CD54 upregulation in only one of the four donors, which,unlike the activity seen with 2141 or BMS-986090, was not dependent onthe addition of CD32a-expressing CHO cells.

Materials and Methods for Examples 1 and 2

FcγR binding SPR: FcγR binding can be measured in vitro using purifiedFcγRs using methods, such as BIAcore™ surface plasmon resonance (SPR).One method tests the binding of purified His-tagged FcγR proteins(FcγR-his) to antibodies that are captured on a sensor surfacecontaining protein A which has been immobilized using standardethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS)chemistry with ethanolamine blocking. These experiments are performed ona BIAcore™ T200 instrument (GE Healthcare, Marlborough, Mass.) at 25° C.For example, samples of purified antibody at 3 μg/ml concentration arefirst captured on the immobilized protein A surface using a 15 second(s) contact time at 10 μl/min flow rate. This is followed by the bindingof purified FcγR-His proteins at various concentrations, such as 10μM-1.5 nM (2:1 dilution series) or 10 μM-13.7 nM (2:1 dilution series),using 120 s association and dissociation times at a flow rate of 30ii/min. All steps are performed in a running buffer consisting of 10 mMNaPO₄, 130 mM NaCl, 0.05% p20 (PBS-T) pH 7.1. FcγR proteins tested inthese studies include the “high affinity” FcγR CD64 (hFcγRI), as well asthe “low affinity” FcγRs CD32a-H131 (FcγRIIa-H131), CD32a-R131(FcγRIIa-R131), CD32b (FcγRIIb), CD16a-V158 (FcγRIIIa-V158), andCD16a-F158 (FcγRIIIa-F158), which were expressed and purified in house.SPR data are fit to either a 1:1 Langmuir model, or a 1:1 steady statemodel using BIAcore™ T200 evaluation software to obtain values for theassociation rate constant (ka), dissociation rate constant (kd) anddissociation constant (K_(D)).

To compare binding responses for different FcγRs, SPR data can beanalyzed by calculating the maximum binding response as a percentage ofthe theoretical maximum binding response (% Rmax), using the equation:% Rmax=(Binding Response Analyte)/[((Mw Analyte)/(Mw Ligand))×(ResponseLigand)×(analyte:ligand stoichiometry)]  EQUATION 1:where “Analyte” is the FcγR and “Ligand” is the captured antibody. Thisanalysis does not take into account the mass of glycosylation ofantibody or FcγR, and assumes 100% fractional activity for the capturedligand. Since the FcγRs are glycosylated, the % Rmax values aretypically great than 100% under saturating conditions.

CD40 binding kinetics and affinity: The monovalent CD40 binding affinityof the antibody molecules is measured by surface plasmon resonance (SPR)on a BIAcore™ T200 instrument (GE Healthcare Life Sciences) at 25° C. or37° C. by capturing antibody on an immobilized protein A sensor chipsurface, and then binding human-CD40-monomer protein (generated inhouse) using, for example, an association time of 180 seconds, anddissociation time of 180 seconds or 360 seconds at 30 μl/min in PBS-T,pH 7.1. SPR data are fit to a 1:1 Langmuir model using BIAcore™ T200evaluation software to obtain values for the association rate constant(ka), dissociation rate constant (kd) and dissociation constant (KD).

Titer analysis: Titer analysis was performed using BiolayerInterferometry (BLI) on an Octet® RED instrument (ForteBio, Freemont,Calif.) at 25° C. Antibodies are captured from supernatant using proteinA sensor tips using association time of 120 seconds and the bindingresponse is measured and compared to a standard curve obtained using acontrol antibody sample to determine the concentration of antibody inthe supernatant.

Primary Cell Isolation and Culture: Peripheral blood mononuclear cells(PBMC) were isolated from heparinized human blood by Ficoll densitygradient separation. Monocytes were isolated from PBMC following theManual EasySep™ protocol (STEMCELL, Vancouver, Canada). One million ofisolated monocytes were plated in in each well of a 6-well plate in 6mLs of complete media (RPMI-1640, 10% heat-inactivated fetal bovineserum, 100 units/ml penicillin-streptomycin), containing IL-4 (100ng/ml) and GM-CSF (100 ng/ml) and incubated for 6 days at 37° C./5% CO₂,changing media every other day and replacing it with fresh mediacontaining the same concentration of cytokines. iDCs (immature dendriticcells) were harvested on day 6, washed thoroughly, and re-suspended incomplete media.

Treatment of iDCs with anti-CD40 Antibodies in the Presence or Absenceof FcγR Clustering/Crosslinking: Titrations of the various biologicalagents were made in complete media, and added to duplicate 96-wellplates. In the case of cross-linking, antibodies were added to the cellsfor 30 min prior to the addition of CD32a-expressing CHO cells at aratio of 1:6. Cells were incubated at 37° C./5% CO₂ for approximately18-20 hours, 150 μL of supernatant was removed from each well, diluted1:5 and evaluated for protein concentrations of IL-6, TNFα and IL-12using a commercially available ELISA kits (R&D Systems, Minneapolis,Minn.), according to manufacturer's instructions. The cells remaining inthe plates from the harvested supernatants were combined into 1 sampleper duplicate treatment, and transferred to new 96-well round bottom(RB) plate, and placed at 4° C. Cells were washed with D-PBS, Ca++ andMg++ free, and stained for 30 min on ice for cell viability using theLIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Carlsbad,Calif.). Cells were washed and re-suspended in D-PBS, Ca++ and Mg++free, 2% FBS, 0.1% NaN₃ (staining buffer) and blocked with 5 μl/well ofHuman TruStain FcX™ (Fc Receptor Blocking Solution, Biolegend, SanDiego, Calif.) in staining buffer.

DCs were immuno-stained with: PerCpCy5.5-conjugated αCD3, αCD19, αCD14(Lin⁻) BUV395-conjugated αCD11c (BD Biosciences, San Diego, Calif.),APC-conjugated αCD86 (Biolegend, San Diego, Calif.), PE-conjugated αCD83(eBioscience, San Diego, Calif.), FITC-conjugated αCD54 (Biolegend, SanDiego, Calif.), and incubated at 4° C. for 45 minutes. Cells were washedtwice in staining buffer and fixed (15 at RT, protected from light), byadding 100 μl of BD Cytofix Fixation Buffer (BD Bioscience, San Diego,Calif.). DCs were evaluated for CD86, ICAM-1 and CD83 expression using aLSRII-Fortessa Flow Cytometer (BD Biosciences, San Diego, Calif.), andFlowJo analysis software (Treestar, Ashland, Oreg.).

Inhibition of CD40L induced human B cell proliferation: Human tonsillarB cells were obtained from pediatric patients during routinetonsillectomy and isolated by mincing and gently mashing the tissue,passing the cells through a screen and isolating mononuclear cells withdensity gradient separation using human Lympholyte®-H separation media(Cedarlane Labs, Burlington, ON). Mononuclear cells were collected fromthe interface, washed, and rosetted with sheep red blood cells (SRBC,Colorado Serum Company; Denver, Colo.) for one hour at 4° C., followedby density gradient separation to remove T cells. Cells were againwashed and re-suspended in RPMI containing 10% FBS (complete media).Titrations of antibodies were made in complete media, and added intriplicate to 96-well round bottom (RB) plates. 1×10⁵ tonsillar human Bcells were added and stimulated with either soluble IZ-hCD40L (2 μg/mL),or with Chinese hamster ovary cells stably transfected with human CD40L(CHO-hCD40L) irradiated with 10,000 rads, and plated at 2×10³cells/well, in a final volume of 200 μL in each well. Plates wereincubated at 37° C./5% CO₂ for 72 hours, labeled for the last 6 hourswith 0.5 μCi of ³[H]-thymidine per well, harvested, and counted byliquid scintillation. B cell proliferation was quantitated based onthymidine incorporation.

Example 3: In Vitro Fc Receptor Assays

Antibodies can exert effector functions, such as complement dependentcytoxicity (CDC), and antibody dependent cellular cytotoxicity (ADCC),by binding of the Fc region to Fc gamma receptors (FcγRs) on the surfaceof immune cells or complement factors. Antibody dependent cellularphagocytosis is another potential Fc effector function. To furthercharacterize the properties of the humanized Y12XX antibodies, theantibodies were assayed for complement dependent cytotoxicity (CDC),antibody dependent cellular phagocytosis (ADCP), and antibody dependentcellular cytotoxicity (ADCC). Table 15 lists the antibodies assayed inthis example.

TABLE 15 Name Reference 1 BMS-986291 (Y1238- See WO 2018/217976hz1-P238K) 2 15B5-hz61-P238K anti-CD40 antibody (produced in house) 35F11-45-P238K See WO 2018/217976 4 Y12XX-hz28-P238K 5 Y12XX-hz40-P238K 6Y12XX-hz42-P238K 7 Antibody C anti-CD40 antibody (See Ristov et al.(2018) Am J Transplant. 18(12): 2895- 2904. Epub 2018 May 24.) 8 BI-LALASee U.S. Pat. No. 9,090,696, heavy chain sequence SEQ ID NO: 32 andlight chain sequence SEQ ID NO: 31; IgG1 isotype containing themutations L234A-L235A 9 BMS-986090 CD40 domain antibody (BMS3h-56-269-IgG₄ Fc fusion polypeptide); see, e.g., WO 2012/145673) 10 TT hIgG1Human anti-tetanus toxin antibody, IgG1 isotype (produced in house)(isotype control) 11 CD20 hIgG1 Human anti-CD20, IgG2 isotype (producedin house) (positive control)

The CDC assay was performed as follows. “CDC Assay Medium” refers toRoswell Park Memorial Institute medium (RPMI)-1640 (HyClone) withL-glutamine, phenol red-free (HyClone) supplemented with 0.1% BSA(Sigma), and 1% Penicillin-Streptomycin (Life Technologies). Fifty (50)microliters of target cells (5×10⁵ cells/mL in CDC assay medium) wereadded to wells of a 96-well assay plate. The target cells were Rajicells which endogenously express CD40 (obtained from ATCC). Serialdilutions (from 133 to 0.002 nM) were prepared for each antibody tested,and 25 microliters of each antibody concentration were added to eachwell. Twenty-five microliters of human complement (obtained from Quidel;diluted 1:3 with CDC assay medium) was added to each well. The assayplates were incubated at 37° C. for 4 hours in a humidified incubator.After the incubation, 100 microliters of CellTiter-Glo® (Promega,Madison, Wis.) was added to each well. Luminescence data was thenacquired with a PerkinElmer EnVision® Plate Reader (PerkinElmer,Waltham, Mass.). Percent viability was calculated relative to isotypecontrol (100% viable). The resulting values are plotted against antibodyconcentration. Percentage of cell viability is plotted for each antibodyusing Prism v5.01 software from GraphPad Inc.

The CDC assay was performed twice. In the second assay, freshly thawedhuman complement serum was used. The results are depicted in FIGS. 3Aand 3B. FIG. 3A depicts the first iteration of the assay, and FIG. 3Bdepicts the second iteration of the assay. CD20 hIgG1 is a positivecontrol and showed cytotoxicity. No detectable CDC activity was presentfor the anti-CD40 antibodies assayed, and specifically, none of thehumanized Y12XX antibodies assayed induced complement-dependentcytotoxicity.

The ADCP assay was performed as follows. “ADCP assay media” refers toRPMI-1640 media with L-glutamine, phenol red-free (HyClone) supplementedwith 10% ultra-low IgG FBS (Gibco). The effector cells were primaryhuman CD14+ monocytes purified from fresh PBMCs from 2 different healthyhuman donors. The target cells were again Raji cells. The Raji cellswere labeled with 2.0 μM PKH26 (red fluorescent dye; Sigma), and theconcentration was adjusted to 4×10⁶ cells/mL in ADCP assay media. Thelabeled target cells were pre-coated with antibodies by adding labeledtarget cells (50 μL/well) to a V-bottom 96-well plate containing 50μL/well of test or control antibody, and incubating for 30 minutes overice. The cells were washed, then effector cells (CD14⁺ monocytes) wereadded (100 μL/well) to result in a final effector cell-to-target cellratio (E:T) of 1:4 and a final antibody concentration ranging from 30 nMto 0.1 nM. The plate was then placed in a humidified 37° C. incubatorfor 1 hour. Cells were stained with APC-anti-CD89 (BioLegend) for 30 minon ice and analyzed by flow cytometer (BD Canto™, BD Biosciences, SanJose, Calif.). Cells were gated for CD89+ cells and subsequently forstained phagocytosed effectors (CD89+, PKH26+). The percentage ofphagocytosis was calculated as the population of CD89+, PKH26+ cellsamong the total CD89+ cells. Background value from the isotype controlwas subtracted to achieve the final percentage of phagocytosis. Data wasanalyzed using FlowJo software and Prism v5.01 software from GraphPadInc.

Exemplary data using CD14⁺ monocytes from two different donors aredepicted in FIGS. 4A and 4B. CD20 hIgG1 is a positive control andinduced phagocytosis of Raji cells, as expected. BMS-986090 also inducedphagocytosis. In contrast, none of the other antibodies tested,including the humanized Y12XX anti-CD40 antibodies of this disclosure,induced detectable phagocytosis in this assay.

The ADCC assay was performed as follows. “ADCC assay media” refers toRPMI-1640 with L-glutamine, phenol red-free (HyClone) supplemented with10% ultra-low IgG FBS (Gibco), and 1 mM sodium pyruvate (LifeTechnologies). Primary human NK (natural kill) cells were purified fromfresh PBMCs from 2 different in-house donors, and used as effectorcells. PBMC were purified from heparinized whole blood samples bydensity gradient centrifugation and washed with PBS supplemented with 2%FBS (HyClone). NK cells were isolated from PBMC by negative selectionusing a magnetic bead-based separation kit (Miltenyi Biotec). Toactivate the NK cells, purified NK cells were resuspended at 1×10⁶cells/mL in MyeloCult H5100 media (StemCell Technologies) supplementedwith 1 μM hydrocortisone (StemCell Technologies) and 500 IU/mLrecombinant human IL-2 (Peprotech) and incubated overnight at 37° C. Thefollowing day, activated NK effector cells were washed twice in ADCCassay media and the concentration was adjusted to 5×10⁵ cells/mL in ADCCassay media. Raji cells (the target cells) were labeled with calcein, asfollows. Calcein AM (Life Technologies) reagent was prepared by adding20 μL of ultrapure DMSO to the reagent tube containing 50 μg oflyophilized reagent. A volume of 2 μL of reconstituted Calcein AM wasadded to the suspended Raji cells for every 1 mL of volume; the cellswere vortexed and placed in a humidified 37° C. incubator for 30minutes. After the incubation period, the labeled target cells werewashed 3 times with ADCC assay media, and the concentration was adjustedto 10⁵ cells/mL in ADCC assay media. Labeled target cells (50 μL/well)were added to a V-bottom 96-well plate containing 50 μL/well of test orcontrol antibody. Activated NK effector cells were then added (100μL/well) to result in a final effector cell-to-target cell ratio (E:T)of 10:1, and a final antibody concentration ranging from 0.0002 to 1μg/mL. The plate was then placed in a humidified 37° C. incubator for 2hours. Supernatant (50 μL/well) was transferred into an optical 96-wellblack plate, and calcein release was measured by reading fluorescenceintensity using an EnVision® Plate Reader (PerkinElmer, Waltham, Mass.)set to 485 excitation and 535 nm emission filters.

Target cells incubated with effector cells in the absence of antibodyprovided the control for background of antibody-independent lysis(spontaneous lysis), while target cells lysed with 20 μL/well 10%Tween-20 lysis buffer represented maximal release in the assay.

The percentage of antibody-dependent cell lysis was calculated based onmean fluorescence intensity (MFI) with the following formula:

$\begin{matrix}{\left( \frac{{{test}\mspace{14mu}{MFI}} - {{mean}\mspace{14mu}{background}}}{{{mean}\mspace{14mu}{maximum}} - {{mean}\mspace{14mu}{background}}} \right) \times 100} & {{Equation}\mspace{14mu} 2}\end{matrix}$Percentage of target cell lysis was plotted for each antibody usingPrism v5.01 software from GraphPad Inc.

Exemplary data using NK cells from two different donors are depicted inFIGS. 5A and 5B. Target cells were killed by positive control anti-CD20antibody. In contrast, ADCC is low-to-negative for all of the anti-CD40antibodies, indicating that these antibodies do not induceantibody-dependent cytotoxicity of Raji cells and evidencing the CD40antagonism of these antibodies.

In summary, in this example, the potential of the humanized Y12XXanti-CD40 antibodies of this disclosure, and specifically,Y12XX-hz28-P238K, Y12XX-hz40-P238K, and Y12XX-hz42-P238K, to mediateADCC (antibody-dependent cellular cytotoxicity), ADCP(antibody-dependent cellular phagocytosis), or CDC (complement-dependentcytotoxicity) was tested using endogenous-CD40-expressing Raji cells astargets. Anti-CD20 antibody was used as a positive control. For ADCC, NKcells were used as effector cells, and two experiments were run witheffector cells from different donors. In each case, none ofY12XX-hz28-P238K, Y12XX-hz40-P238K, and Y12XX-hz42-P238K induced lysisof Raji cells. None of Y12XX-hz28-P238K, Y12XX-hz40-P238K, andY12XX-hz42-P238K induced CDC of Raji cells beyond the effect of humanIgG1 isotype control. For ADCP, CD14+ monocytes were utilized aseffector cells, and in this system none of Y12XX-hz28-P238K,Y12XX-hz40-P238K, and Y12XX-hz42-P238K promoted cellular phagocytosis.In contrast, BMS-986090 exhibited cellular phagocytosis.

Example 4: Assay of NF-kB/AP-1 Signaling

The objective of this example was to assess the NF-κB/AP-1 inducibleSEAP (secreted embryonic alkaline phosphate) activity on Ramos-Blue™Cells (InvivoGen) resulting from stimulation with anti-CD40 antibodies.

Ramos-Blue™ Cells are a human B lymphocyte reporter cell line thatexpress an NF-κB/AP-1 inducible secreted embryonic alkaline phosphate(SEAP) reporter gene. The Ramos-Blue™ cell line has been used forNF-κB/AP1 signaling as well as in Toll-like Receptors' (TLR's) signalingpathways, Ramos-Blue™ Cells endogenously express CD40 and are responsiveto CD40 and TLR. When Ramos-Blue™ Cells lines are stimulated, theyproduce SEAP in the cell culture supernatant. SEAP can be detected byusing the QUANTI-Blue™ detection medium (InvivoGen, San Diego, Calif.).Levels of SEAP can be observed visually or by using a spectrophotometerat 620 nm. Ramos-Blue™ Cells do not express CD32 (FcγRII).

Table 6 lists the test materials (antibodies and other polypeptide)assayed in this example. All of the test materials were prepared inhouse by BMS.

TABLE 16 Test Materials Description BMS-986325 Y12XX-hz28-P238K (fullyhuman anti-CD40 isotype hIgG (P238K) mAb BMS-986090 fully humananti-CD40 domain antibody IgG4 Fc fusion protein mAb 134-2142 fullyhuman anti-CD40 (CD40-2142 (hIgG2-Fc)) monoclonal antibody (agonist)Anti-DT1D12-B16F7-hIgG1.3f mAb Negative Control CD40L-IZ a humanCD40L-Trimer (h-IZ-hCD40L-Trimer)

Ramos-Blue™ Cells were purchased from InvivoGen. For this assay, atransduced cell line designated herein as Ramos Blue Cells #4, wasprepared by transducing Ramos-Blue™ Cells with human CD32 (FcγRII). Bothtypes of Ramos-Blue™ cells were cultures in Ramos Cell Culture Medium(Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FetalBovine Serum (FBS), 2 mM L-Glutamine, penicillin and streptomycin (100U/mL, −100 ug/mL), 100 ug/mL of Normomicin, and Zeocin as drug selection(100 ug/mL)). Cells were cultured at 37° C. in 5% CO₂. Both types ofcells were passed every 3 days and maintained at a cell density of0.5×10⁶ cell/ml. Cells were used until cell passage #21 (P21). AfterP21, the cells were discarded.

The assay was performed as follows. “AIM V™ medium” refers to serum freemedium supplemented with L-glutamine, 50 μg/mL streptomycin sulfate, 10μg/mL gentamicin sulfate (Thermo Fisher Scientific). To assess CD40agonist activity by CD40 antagonist antibodies on the NF-kB/AP-1activity in Ramos Blue Cells #4, cells were washed twice (2×) with AIMV™ medium without antibiotics Normocin/Zeocin. The cells were thencentrifuged at room temperature for 10 minutes at 2,000 rpm. Medium wasaspirated carefully to not disrupt the cell pellet. One (1) mL of AIM V™was added to the cell pellet to re-suspend the cell pellet, and anadditional 9 ml of AIM V™ was added after resuspension. Cells werecounted using the cell counter by adding 20 microliters (μl) ViaStain™AOPI (acridine orange/propidium iodide) staining solution (NexcelomBioscience, LLC, Lawrence, Mass.) and 20 μl of Ramos-Blue™ Cellssuspension.

Ramos-Blue™ Cells were adjusted to 4×10⁶ cell/mL in AIM V™ serum freemedium. One hundred (100) μl of 400K Ramos-Blue™ Cells were added perwell in a flat bottom tissue culture plate. Then, 100 μl of BMS-986325,BMS-986090, mAb134-2141 (CD40-2142), or control was added to eachcorresponding well. CD40L-IZ was used as positive control. Ramos-Blue™cells (0.4×10⁶ cells/well) in AIM V™ were included as negative controlfor the assay. The final volume was 200 μl/well.

Plates were incubated at 37° C. in a 5% CO₂ incubator for 20 hours.After 20 hours of cell culture, plates were centrifuged for 10 minutesat 2000 rpm.

Forty (40) μl of cell culture supernatant from stimulated Ramos cellswere added to wells of a flat bottom plate. Then, 160 μl of QUANTI-Blue™Solution was added per well. The final volume was 200 μl/well.

Plates were incubated at 37° C. in a 5% CO₂ incubator for 1 to 6 hours.SEAP levels were measured every 60 minutes at 620 nm using the EnVision®Reader Optical (OD: 620 nm).

Exemplary data are depicted in FIG. 6. In this example, anti-CD40monoclonal antibodies were tested against Ramos-Blue™ Cells lacking CD32or Ramos-Blue™ Cells transduced with CD32 (Ramos Blue #4) by assessingthe NFκ-B/AP-1 inducible SEAP activity on Ramos-Blue™ Cells uponstimulation with anti-CD40 antibodies.

Addition of CD40-2142 induced a significant signaling response in thisassay FIGS. 6A and 6 b). These results indicate that CD40-2142 is a fullagonist in this assay system. Addition of BMS-986090 showed no responseby using the Ramos-Blue™ Cells (−CD32) (FIG. 6A), but displayed apartial agonism in the assay using the Ramos Blue Cells #4 (Ramos-Blue™Cells transduced with CD32) (FIG. 6B). This result indicates FcγRdependency mediates the agonistic response induced by addition ofBMS-986090. The control polypeptide, trimer CD40L-IZ, induced a responsein both assays as reflected in FIGS. 6C and 6D.

In contrast, addition of BMS-986325 did not induce a significantNFκ-B/AP-1 response using either Ramos-Blue™ Cells (CD32⁻) (FIG. 6A) orRamos #4 (CD32⁺) (FIG. 6B). These data indicate that BMS-986325 did notagonize CD40 and did not engage CD32 (FcγRII). These data support thatreduced engagement of low affinity FcγRs, such as CD32 (FcγRII), reducesthe likelihood of undesirable agonist signaling and undesirablepotential for toxicity.

Example 7: Summary of Non-Clinical Pharmacokinetics Evaluation ofBMS-986325

The pharmacokinetics (PK) of BMS-986325 (Y12XX-hz28-P238K) wereevaluated in mice and cynomolgus monkeys. Since BMS-986325 does notcross react to murine CD40 receptors, the PK evaluated in mice isintrinsic or non-specific PK. BMS-986325 cross reacts with monkey CD40receptors, therefore the total PK (specific and non-specific PK) wasevaluated in monkeys. After intravenous (IV) administration ofBMS-986325 (single 1- and 10-mg/kg doses) to mice, BMS-986325 exhibitedlow total serum clearance “CLT” of 0.5 to 1.02 mL/d/kg, limited volumeof distribution at steady state “Vss” of 0.12 to 0.19 L/kg, and longapparent elimination half-life “T-HALF” of 118 to 183 hours (˜5 to 8days).

In monkeys, a single subcutaneous (SC) dose of BMS-986325 wasadministered. The dose administered is a dose at which specificclearance (target-mediated drug disposition “TMDD”) is not saturated.After the single SC dose, BMS-986325 was well absorbed, with an absolutebioavailability of 70.4% (relative to exposures at the same IV dose).After IV administration of BMS-986325 (10 mg/kg single dose) to monkeys,BMS-986325 exhibited a CLT of 0.41 mL/d/kg, a limited Vss of 0.05 L/kg,and a T-HALF of 100 hours (˜4 days). The time to maximum plasmaconcentration “Tmax” following a single SC dose of BMS-986325 (doses of1, 10, and 100 mg/kg administered) to monkeys was 24 to 54 hours. Therewere more-than-dose-proportional increases in exposure (maximumconcentration “Cmax” and area under the concentration vs time curveextrapolated from time zero to infinity “AUC[INF]”) and an increase inT-HALF with dose (˜31, ˜119, and ˜197 hours at 1, 10, and 100 mg/kg,respectively). These data suggest nonlinear PK and a saturable clearancemechanism; this likely results from target (CD40)-mediated clearance,reflecting TMDD. In this single-dose PK study, anti-drug antibody (ADA)formation was detected in ˜50% of monkeys, but had no apparent impact onthe overall PK parameters.

Pharmacokinetic/pharmacodynamic modeling (TMDD model with quasisteady-state assumption [TMDD-Qss]) was used to describe the nonlinearPK observed in monkeys, establish a relationship between serum drugexposure and CD40 receptor occupancy (RO) and subsequent human doseprojection.

Although the present embodiments have been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of these embodiments, andwould readily be known to the skilled artisan.

These and other aspects disclosed herein, including the exemplaryspecific treatment methods, medicaments, and uses listed herein, will beapparent from the teachings contained herein.

The invention claimed is:
 1. An isolated antibody, or antigen binding portion thereof, that specifically binds to human CD40, wherein said antibody comprises a first polypeptide portion comprising a heavy chain variable region, and a second polypeptide portion comprising a light chain variable region, wherein: said heavy chain variable region comprises a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ ID NO: 3; and said light chain variable region comprises a CDR1 comprising SEQ ID NO: 7, a CDR2 comprising SEQ ID NO: 8, and a CDR3 comprising SEQ ID NO:
 9. 2. The isolated antibody or antigen binding portion thereof of claim 1, wherein said antibody or antigen binding portion thereof antagonizes a CD40 activity.
 3. The isolated antibody or antigen binding portion thereof of claim 1, wherein: said heavy chain variable region comprises a CDR1 consisting of SEQ ID NO: 1, a CDR2 consisting of SEQ ID NO: 2, and a CDR3 consisting of SEQ ID NO: 3; and said light chain variable region comprises a CDR1 consisting of SEQ ID NO: 7, a CDR2 consisting of SEQ ID NO: 8, and a CDR3 consisting of SEQ ID NO:
 9. 4. The isolated antibody or antigen binding portion thereof of claim 1, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4, and said light chain variable region comprises the amino acid sequence of SEQ ID NO:
 10. 5. The isolated antibody or antigen binding portion thereof of claim 1, wherein said first polypeptide portion comprises a human heavy chain constant region; and said second polypeptide portion comprises a human light chain constant region.
 6. The isolated antibody or antigen binding portion thereof of claim 5, wherein said human heavy chain constant region is a human IgG1 Fc domain comprising a mutation at Kabat position 238 that reduces binding to Fc-gamma-receptors (FcγRs), wherein proline 238 (P238) is substituted with an amino acid selected from the group consisting of: lysine, serine, alanine, arginine, and tryptophan, and wherein the antibody or antigen binding portion thereof has reduced FcγR binding.
 7. The isolated antibody or antigen binding portion thereof of claim 6, wherein the substituted amino acid is lysine.
 8. The isolated antibody or antigen binding portion thereof of claim 7, wherein the Fc domain comprises an amino acid sequence selected from: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:
 29. 9. The isolated antibody or antigen binding portion thereof of claim 6, wherein: said heavy chain variable region comprises a CDR1 consisting of SEQ ID NO: 1, a CDR2 consisting of SEQ ID NO: 2, and a CDR3 consisting of SEQ ID NO: 3; and said light chain variable region comprises a CDR1 consisting of SEQ ID NO: 7, a CDR2 consisting of SEQ ID NO: 8, and a CDR3 consisting of SEQ ID NO:
 9. 10. The isolated antibody or antigen binding portion thereof of claim 9, wherein the human IgG1 Fc domain comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO:
 23. 11. The isolated antibody or antigen binding portion thereof of claim 1, wherein the first polypeptide portion comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 30, and SEQ ID NO: 31; and the second polypeptide portion comprises or consists of the amino acid sequence of SEQ ID NO:
 11. 12. The isolated antibody or antigen binding portion thereof of claim 1, wherein the first polypeptide portion comprises or consists of the amino acid sequence of SEQ ID NO: 5; and the second polypeptide portion comprises or consists of the amino acid sequence of SEQ ID NO:
 11. 13. The isolated antibody or antigen binding portion thereof of claim 1, wherein the isolated antibody or antigen binding portion thereof is humanized.
 14. The antibody or antigen binding portion thereof of any of claim 1, wherein the antigen binding portion thereof is an scFv-Fc.
 15. The antibody or antigen binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof is linked to a therapeutic agent.
 16. The antibody or antigen binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof is linked to a second functional moiety having a different binding specificity than said antibody or antigen binding portion thereof.
 17. The antibody or antigen binding portion thereof of claim 1, further comprising an additional moiety.
 18. The isolated antibody or antigen binding portion thereof of claim 1, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 4, and said light chain variable region comprises the amino acid sequence of SEQ ID NO:
 16. 19. A pharmaceutical composition comprising: a) the antibody, or antigen binding portion thereof, of claim 1; and b) a pharmaceutically acceptable carrier.
 20. A method of treating or preventing an immune response in a subject comprising administering to the subject the antibody, or the antigen binding portion thereof, of claim
 1. 21. A method of treating or preventing an autoimmune or inflammatory disease in a subject, comprising administering to the subject the antibody, or the antigen binding portion thereof, of claim
 1. 22. The method of claim 21, wherein the antibody, or the antigen binding portion thereof, is administered with an immunosuppressive, immunomodulatory, and/or anti-inflammatory agent.
 23. The method of claim 22, wherein said immunosuppressive, immunomodulatory, and/or anti-inflammatory agent is CTLA4 mutant molecule L104EA29Y-Ig (belatacept).
 24. The method of claim 21, wherein the subject has a disease selected from the group consisting of: Addison's disease, allergies, anaphylaxis, ankylosing spondylitis, asthma, atherosclerosis, atopic allergy, autoimmune diseases of the ear, autoimmune diseases of the eye, autoimmune hepatitis, autoimmune parotitis, bronchial asthma, coronary heart disease, Crohn's disease, diabetes, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, idiopathic thrombocytopenic purpura, inflammatory bowel disease, an immune response to recombinant drug products, lupus nephritis, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, transplant rejection, vasculitis, and ulcerative colitis.
 25. The method of claim 21, wherein the subject has Sjogren's syndrome. 